MX2013005485A - Drought tolerant plants. - Google Patents

Abstract

The present specification teaches the generation of drought tolerant plants. The present disclosure enables manipulation of a phenotypic characteristic referred to herein as "stay-green" to generate drought tolerant plants by recombinant, mutagenic and/or breeding and selection methods. Plant management practice systems to increase crop yield and harvest efficiency in water-limited environments are also taught herein.

Description

TOLERANT PLANTS TO LA MARCHITEZ Presentation Data The present application is associated with US Provisional Application No. 61 / 413,902, filed on November 15, 2010, which is incorporated in its entirety to the present invention as a reference.

Field of the Invention The present specification teaches the generation of plants tolerant to wilting. The present description allows the manipulation of a phenotypic characteristic referred to herein as "ever green" to generate plants tolerant to wilting through recombinant, mutagenic and / or seeding and selection methods. Systems to practice plant management are also taught to increase crop production and harvest efficiency in environments with limited water.

Background of the Invention The bibliographic details of the publications referred by the author in the present specification are presented in alphabetical form at the end of the description.

The reference to any prior art in the present specification shall not be taken as an acknowledgment or any form of suggestion that said prior art forms part of the general common recognition in any country.

A growing human population needs increases in crop productivity. This has been an important goal for plant breeders and plant breeders. One method to improve crop productivity involves the selection of plant traits that facilitate greater grain yield and stability (Springer, Nature Genetics 42: 475-476, 2010). This method has been referred to as the "Green Revolution". Other methods include the development of ideal plant architectures that have led, for example, to the identification of a quantitative trait locus (QTL) that encodes a squamous promoter-type 14 binding protein (OsSPL14) in rice, and that facilitates improved rice production (Jiao et al., Nature Genetics 42: 541-544, 2010; Miura et al., Nature Genetics 42: 545-549, 2010).

Wilt is the most important restriction in cereal production worldwide. Sorghum is a storehouse of wilt resistance mechanisms, including C4 photosynthesis, deep roots and thick wax on the leaf, which allow growth in hot and dry environments. Tolerance to wilting makes sorghum especially important in dry regions, such as sub-Saharan Africa, central western India, northeastern Australia and the southern plains of the United States of America. With increasing pressure on the availability of water resources scarce, it becomes increasingly important to identify traits associated with grain production under wilting conditions.

The mechanism of wilt adaptation identified in sorghum, which results in the retention of green leaves for longer periods during grain formation, is known as "evergreen". The "evergreen" mechanism has been associated with high grain production under wilting after flowering in sorghum (Borrell et al., Crop Sci. 40: 1037-1048, 2000b, Kassahun et al., Euphytica 72: 351 -362, 2010), wheat (Triticum aestivum L.) [Spano et al, J. Exp. Bot. 54: 1415-1420, 2003; Christopher et al, Aust. J. Agrie. Res. 59: 354-364, 2008], rice (Oryza sativa L.) [ashiwagi et al, Plant Physiology and Biochemistry 44: 152-157, 2006] and corn (Zea mays L.) [Zheng et al, Plant Breed 725: 54-62, 2009]. In addition, the production of the grain under wilting can be indirectly affected, improving the resistance to cork rot (Macrophomina phaseolina [Tassi] Goid.) (Tenkouano et al, Theor, Appl. Genet, 85: 644-648, 1993, Garud et al. al, Int. Sorghum and Millets Newsl, 43: 63-65, 2002). This reduces fixation (Reddy et al, Euphytica 759: 191-198, 2008), allowing plant breeders to exploit the positive association between plant height and grain production (Jordán et al, Theor. Appl. Genet 70 (5: 559-567, 2003) The "always green" mechanism has been a selection criterion important for sorghum cultivation programs that address wilt adaptation in both the United States of America (Rosenow et al, Agrie Water Manag. 7: 207-222, 1983) and Australia (Henzell et al. Australia Int. Sorghum and Millets News, 38: 1-9, 1997).

A body of considerable physiological evidence is being mounted as a support for this mechanism (Borrell et al, Crop Sci. 40: 1026-1037, 2000a, Borrell and Hammer, Crop Sci. 40: 1295-1307, 2000; Harris et al, J. Exp. Bot. 58: 327-338, 2007, Christopher et al, 2008 supra, Van Oosterom et al, Field Crops Res. 175: 19-28, 2010a and Van Oosterom et al, Field Crops Res. 115: 29 -38, 2010b). Although this mechanism of resistance to wilt has been used by sorghum creators in the United States of America and Australia for 25 years, and the broader physiological basis of the mechanism has been better understood, the mechanisms of origin remain unknown until now. and the genetic place involved.

Under conditions of water limitation, grain production is a function of transpiration (T), transpiration efficiency (TE), and harvest index (Hl) [Passioura, J. Aust. Inst. Agrie. Sci. 43: 117-120, 1977]. Within this framework, grain production is linked to the subsequent outcrop T (Turner, J. Exp. Bot. 55: 2413-2425, 2004; Manschadi et al, Fund Plant, Biol 53: 823-837, 2006), to which Hl increases with the fraction of the total T culture used after flowering (Passioura, 1977 above, Sadras and Connor, Field Crops Res. 26: 227-239, 1991, Hammer, Agrie, Sci. J 9: 16-22, 2006). The subsequent flowering T increased, is associated with a reduced wilting tension around flowering, which can positively affect the growth rate of the crop in the flowering of cereals, and therefore the number of grains (Andrade et al, Crop Sci. 42: 1173-1179, 2002; Van Oosterom and Hammer, Field Crops Res. 108: 259-268, 2008). If the total amount of water available is limited, the subsequent flowering T may be increased by restricting the previous flowering T. This can be achieved by restricting the size of the cover, either genetically or through crop management. However, a smaller cover will only reduce the total T if your TE is not compromised. Significant genotypic differences in TE have been reported for sorghum (Hammer et al, Aust J. Agrie Res. 48: 649-655, 1997, Henderson et al., Aust. J. Plant Physiol. 25: 111-123, 1998; Mortlock and Hammer, J. Crop Prod. 2: 265-286, 1999; Xin et al, Field Crops Res. 111: 74-80, 2009). Alternatively, water after flowering can be increased by increasing the amount of total water to which the crop has access, either through deeper rooting, or a lower lower limit of water extraction (Manschadi et al. 2006 above).

The 'always green' mechanism affects a number of the previous processes in sorghum. First, the mechanism 'always green 'reduces the time of water during the period before flowering, restricting the size of the cover (by means of reduced rods and smaller leaves).

Second, the 'always green' mechanism improves water accessibility, increasing the root: shoot ratio. There is some experimental evidence for better water extraction in evergreen lines, although more research is required. These root responses can also be explained by increased auxin transport (Wang et al, Molecular Plant 2 (4): 823-831, 2009). Third, the 'always green' mechanism increases the greenness of the leaves in flowering, effectively increasing the photosynthetic capacity, and therefore, TE (provided that photosynthesis increases proportionally than conductance). The increase in greenness of the leaf is an indirect effect of the reduced leaf mass, for example, nitrogen that is concentrated in the leaf.

The production of more food with less water is one of the main challenges that humanity is currently facing. There is a real and urgent need in both developing and developed countries to identify the genes and genetic networks that control the adaptation to wilting in crop plants. This allows an adaptation to increased wilting in a wide range of crop species grown in environments with limited water worldwide.

Brief Description of the Invention The present disclosure teaches quantitative trait loci (QTLs) associated with and / or which otherwise facilitate the 'always green' phenotype. The QTLs are referred to in the present invention as "always green (Stg) X" where X is an increment number from 1, which represents the region on a chromosome associated with the ever green phenotype.

As taught here, QTLs identify genetic regions in sorghum, which carry loci encoding one to a number of proteins or regulatory agents such as microRNAs, which facilitate the 'always green' phenotype. The expression modulation of one or more of these loci in a crop plant generates a cover architecture that facilitates a change in water use through the plant in the post-flowering period, or an access capacity of increased water during crop growth or increased transpiration efficiency, thereby increasing the harvest index (Hl) and the production of the grain under conditions with water limitation.

In one embodiment, the loci encodes a PIN protein that is associated with an auxin. PIN proteins are auxin efflux transporters that contain transmembrane domains and are located primarily in plasma membranes. The term "PIN" is derived from "pin-type inflorescence".

The term "SbPINn" is used to describe this gene in sorghum, where n is a number that defines the transporter component of auxin efflux, and n is 1 and 11. The reference to "SbPINn" includes its homologs and orthologs in other plants. Examples of SbPINn loci in sorghum include those described in Table 1A, such as but not limited to SbPIN4 and SbPIN2 or their equivalents in other plants. The modulation of expression of a PIN or the expression of a PIN with a particular polymorphic variation is taught in the present invention to facilitate the display of the 'always green' phenotype. The present description teaches PINs of other plants, such as rice. The subscript with letter that has a PIN, specifies its source (for example, Sb, sorghum, Os, rice, etc). The location of a SbPIN in sorghum is determined by the gene ID number (See table 1A). As examples, SbPIN4 corresponds to OsPIN5 and SbPIN2 corresponds to OsPIN3a.

SbPIN4 and SbPIN2 are examples of SbPINs taught here, responsible for the 'always green' trait in sorghum, associated with Stg1, and Stg2, respectively, which results in a range of phenotypes that confer wilting adaptation through decreased water use until flowering (due to reduced stems and smaller leaves), increased access to water (due to the increased root: shoot ratio), increased transpiration efficiency under a moderate water deficit (due to a higher concentration of nitrogen in the leaf), increased biomass per leaf area under the terminal water deficit (due to increased transpiration per leaf area) and increased grain production, grain size and resistance to fixation . Table 1A describes other examples, and includes their equivalents in other plants.

The present disclosure teaches a method for generating a genetically modified plant that uses water more efficiently than a non-genetically modified plant of the same species, wherein the method comprises modulating the expression level of an existing inflorescence locus or pin type introduced. (PIN) in all the tissue or tissue selected in the plant to facilitate an 'always green' phenotype, where the phenotype includes a change in the use of water in the period after flowering, or an increased water capacity during the crop growth or increased transpiration efficiency that results in an increased crop yield and grain yield under conditions with water limitation.

In the present invention, a method for generating a genetically modified plant that uses water more efficiently than a non-genetically modified plant of the same species is enabled, wherein the method comprises introducing a genetic agent into a plant or progenitor of the plant. that encodes a selected PIN protein of SbPINI to 11, or an equivalent thereof of another plant or functional orthologous homolog thereof; or that modulates the expression of a native PIN protein; wherein the level and location of the PIN expression facilitates an 'always green' phenotype, where the phenotype includes, among other things, a cover architecture that facilitates a change in the use of water in the post-flowering period, or an increased water access capacity during crop growth, resulting in an increased grain yield and crop yield under conditions with water limitation.

The present disclosure further teaches a method for generating a genetically modified plant that uses water more efficiently than a non-genetically modified plant of the same species, wherein the method comprises introducing into a plant or progenitor of the plant, a genetic agent that encodes a sorghum PIN protein selected from SbPIN4 and SbPIN2 or an equivalent in another plant, or that modulates the expression of a native PIN. The reference to modulation includes increasing or decreasing the level of expression. In addition, a PIN having a desired expression profile can be selected throughout the tissue or in selected tissues in a plant.

In the present invention a method is enabled to generate a genetically modified plant that uses water more efficiently than an unmodified plant genetically of the same species, wherein the method comprises introducing into the plant or progenitor of the plant, a genetic agent that encodes a product that is associated with, or facilitates the 'always green' phenotype, wherein the phenotype includes a change in the use of water in the post-flowering period, or an improved water access capacity during crop growth or increased transpiration efficiency that results in increased grain yield and crop yield under water limiting conditions, and where the product is selected from the list consisting of SbPINI to 11 or an equivalent thereof in another plant, or where the agent modulates the expression of a native PIN in the plant.

Also taught in the present invention is a method for generating a genetically modified plant that uses water more efficiently than a non-genetically modified plant of the same species, wherein the method comprises introducing into a plant or progenitor of the plant, an agent A gene encoding a protein selected from the list consisting of SbPIN4 and SbPIN2 or an equivalent thereof in another plant, or wherein the agent modulates the expression of a native PIN in the plant. The term "enter" includes by recombinant intervention, as well as by mutagenesis or breeding protocol, followed by selection.

The present description instructs with respect to a method to generate a genetically modified plant, which uses water more efficiently than a non-genetically modified plant of the same species, wherein the method comprises introducing into a plant and / or progenitor of the plant, a genetic agent that codes two or more PIN proteins, or that modulates an expression of two or more native PIN proteins in a plant. Examples of sorghum PINs include SbPINI to 11, such as SbPIN4 and SbPIN2 or an equivalent thereof from another plant. For example, SbPIN4 corresponds to OsPIN5 and SbPIN2 corresponds to OsPIN3a. SbPINs and OsPINS are defined in table 1 A.

Genetically modified plants and their progeny, which exhibit the 'always green' mechanism, are also enabled in the present invention, as well as seeds, fruits and flowers and other reproductive or propagating material.

The genetic material that encodes a PIN protein or functional homologue or orthologous thereof, that is associated with, or that facilitates an 'always green' phenotype, wherein the phenotype includes a shell architecture that facilitates a change in the use of water in the post-flowering period, or an increased water access capacity during crop growth or increased transpiration efficiency, results in increased grain yield and yield index under water limiting conditions, and an agent that modulates the expression of PIN; both enabled in the present description.

In one example, the genetic material is selected from (i) an agent that encodes SbPIN4; and (ii) an agent that modulates the expression levels of SbPIN4 or its equivalent in another plant. In another example, the genetic material selected from (i) an agent encoding SbPIN2; and (ii) an agent that modulates the expression of SbPIN2 or its equivalent in another plant.

The genetic material includes an agent that encodes a SbPIN4 or SbPIN2 protein or a functional homolog or ortholog thereof thereof or an equivalent thereof in another plant that is associated with, or that facilitates an 'always green' phenotype, wherein the phenotype includes a roof architecture that facilitates a change in water use in the post-ering period or an increased water accessibility during crop growth or increased transpiration efficiency that results in a harvest or production rate grain increased under conditions with water limitation; and an agent that modulates the expression of SbPIN4 or SbPIN2, or an equivalent thereof in another plant.

In the present invention a plant management system is taught to reduce the water dependence of the crop, or to otherwise improve the efficiency of water use and to increase the production of the grain or product. The management system of the plant includes the generation of a crop adapted to wilting, which includes cereal plants, using the selection and expression modulation of a PIN locus or its functional equivalent, as defined in the present invention alone or in combination with the introduction of other useful features, such as grain size, root size, tolerance to salt, herbicide resistance, resistance to pests and the like. As an alternative or additionally, the plant management system comprises the generation of plants adapted to wilting and agricultural procedures such as irrigation, nutrient requirement, density and crop geometry, weed control, insect control, aerated soil , reduced rods, raised beds and the like. Examples of a PIN locus include SbPIN1 to 11 (table 1A), such as SbPIN4 and SbPIN2 and one equivalent thereof, in another plant.

The present invention also teaches a business model for crop production with economic yields, wherein the model comprises generating crop plants that have a cover architecture that facilitates a change in the use of water through the plant in the post-ering period, or an increased water access capacity during the growth of the crop, or increased transpiration efficiency, to thereby increase Hl, and the production of grain under conditions with water limitation, to obtain seeds of the generated crop plant, and distribute the seeds to the grain producers for increase production and profits In the present specification the following abbreviations are used: Cl, accounting interval CWU, use of crop water DW, dry weight GLA, green leaf area HD, high density Hl, harvest index HT, high level shank HW, high level water HWHD, high level water, high density (intermediate water tension) HWLD, high level water, low density (less water in tension) LA, area of the leaf LD, low density LT, low level offshoots LW, low water level LWHD, low water level, high density (most of the water stressed) LWLD, low water level, low density (intermediate water tension) NIL, almost isogenic line OsPIN, rice pin PAB, biomass after ering PASM, stem mass after ering PIN, pin-type inescence PPBR, proportion of biomass pre: post-ering QTL, quantitative trait locus ROS, shelter against the rain RWC, relative water content SbPIN, sorghum pin SLW, specific sheet weight SML, statistical machine learning Stg, 'always green' T, Transpiration T2, stem in the axilla of leaf 2 T3, stem in the axilla of leaf 3 T4, stem in the axilla of leaf 4 T5, stem in the axilla of leaf 5 T6, stem in the axilla of leaf 6 TE, transpiration efficiency TS terminal voltage VPD, vapor pressure deficit WW, well wet Table 1A provides information regarding sorghum and rice PIN.

OI or I heard OI Table 1A Details QTL Stg of Sorgo in or in Brief Description of the Figures Some of the figures contain colored representations or entities. Color photographs are available in Patent upon request, or in an appropriate Patent Office. A fee payment may be required if obtained from a Patent Office.

Figure 1 is a graphical representation showing the relation between rootlets per m2 and area of green leaves in flowering, in a range of almost isogenic lines containing various Stg introgressions.

Figure 2 is a graphical representation showing the relationship between rootlets per m2 and green leaf area in flowering, in a range of Stg introgressions in a RTx7000 background grown under two cultivation densities (LD = 10 plants / m2; HD = 20 plants / m2).

Figure 3 is a graphical representation showing the histogram of anticipated values of rootlets per plant in the population of fine mapping Stg1 averaged over three stations.

Figure 4 is a tabulated representation showing a histogram of rootlets per plant in 44 DAE for five genotypes grown under two water regimes. The genotypes comprise RTx7000 (recurrent origin), 6078-1 (donor origin), and three selections from the population of fine mapping Stg1. HWLD = high water content, low density (10 plants / m2). LWLD = low water content, low density (10 plants / m2).

Figure 5 is a graphical representation showing the phenotypic variation in the population of fine mapping Stg1 for the presence of T2.

Figure 6 is a graphical representation showing the phenotypic variation in the population of fine mapping Stg1 for the presence of T3.

Figure 7 is a representation showing a histogram of the presence of T2 for eight high-level stem recombinants and eight low-level stem recombinants from the fine mapping population Stg1.

Figure 8 is a representation showing a histogram of the number of total shoots per plant for five high-level scion recombinants and three low-level sire recombinants from the fine mapping population Stg1. A value of 2.5 was chosen as the arbitrary cut between high and low level scions.

Figures 9A to 9D are graphical representations showing the size distribution of the main sheet of RTX7000 and 6078-1 (Stg7 NIL), VPD.

Figure 10 is a graphical representation showing the distribution of the main sheet of RTx7000, 6078-1 (Stg1 NIL) and three recombinants from the population of fine mapping Stg1 grown under conditions of water limitation and high density in the field (Fig. HD = 20 plants / m2).

Figure 11 is a graphical representation showing the leaf size distribution (L1-6) for the progenitors of the population of fine mapping Stg 1 shown in an igloo.

Figure 12 is a graphical representation showing the leaf length distribution (L1-6) of the progenitors of the fine mapping population Stg1 grown in an igloo.

Figure 13 is a graphical representation showing the width distribution of the leaf (L1-6) of the progenitors of the fine mapping population Stg1 grown in an igloo.

Figure 14 is a graphical representation showing the leaf size distribution (11-11) of the progenitors of the fine mapping population Stg1 grown in an igloo.

Figure 15 is a graphical representation showing the leaf length distribution (L1-10) of the origins of the fine mapping population Stg1 grown in an igloo.

Figure 16 is a plot showing a phenotypic variation histogram of length L10 in a subset of the fine mapping population Stg1 grown in an igloo.

Figure 17 is a diagrammatic representation showing the increased availability of water in flowering that is achieved by the use of reduced water due to two mechanisms (reduced shoots and smaller leaves) in plants containing the Stg1 region.

Figure 18 is a representation showing that the Cover size is modulated by both constitutive and adaptive responses controlled by a gene (s) in the Stg 1 region.

Figure 19 is a graphical representation showing the size distributions of the main sheet of RTx7000, 6078-1 (Stg1 NIL) and three recombines from the population of fine mapping Stg1 grown under conditions with water limitation and high density in the field (HD = 20 plants / m2).

Figure 20 is a graphical representation showing the relationship between the ratio between the leaf area 12 and the total area of the green leaf at flowering for the two parents (6078-1 and RTx7000), and three recombinants from the population of fine mapping Stg1.

Figure 21 is a graphic representation showing the relationship between the total area of the green leaf (cm2 / m2) and the use of water for culture (mm) in flowering for the two parents (6078-1 and RTx7000) and three recombinants from the population of fine mapping Stg1.

Figure 22 is a graphical representation showing the relationship between the area of the green leaf and the use of water (T) in four QTL Stg, and the recurrent parent (RTx7000) in studies of lysimetry under two VPD levels.

Figure 23 is a graphical representation showing a histogram of phenotypic variation for the "root: shoot ratio" L6 in the population of fine mapping Stg1 grown in a igloo.

Figure 24 is a graphical representation showing the temporary pattern of use of cumulative culture water for RTx7000 and Stg1 grown under low density and low density water conditions (20 plants / m2). The vertical line indicates flowering.

Figure 25 is a graphical representation showing the relationship between the length (mm) and greenness (SPAD) of sheet 10 in the fine mapping population Stg1 grown in an igloo.

Figure 26 is a graphical representation showing the relationship between leaf greenness (SPAD) and leaf photosynthesis in a subset of lines of the fine mapping population Stg1, including the parents.

Figure 27 is a graphical representation showing the relationship between leaf greenness (SPAD) and WUE (Liquor) in a subset of lines of the population of fine mapping Stg1, including the parents.

Figure 28 is a graphical representation showing the relationship between leaf greenness (SPAD) and WUE (Liquor) in four Nils Stg (Stg1, Stg2, Stg3 and Stg4) and the recurrent parent (RTx7000).

Figure 29 is a graphical representation showing the relationship between transpiration per leaf area and transpiration efficiency in four QTL Stg and the recurrent parent (RTx7000) in lysimetry studies under two VPD levels.

Figure 30 is a graphical representation showing the relationship between CWU (mm) before and after flowering, in a subset of lines of the population of fine mapping Stg1, including parents, grown under high density conditions (HD) and low density (HD).

Figures 31A and 31B are graphical representations showing the progenitors of cumulative water use for Stg 1 and RTx7000 grown under LWHD and conditions.

Figure 32 is a graphical representation showing the relationship between CWU (mm) before and after flowering in four QTL Stg and the recurrent parent (RTx7000) grown under low water (LW) and low density (LD) conditions ).

Figure 33 is a graphic representation showing the relationship between PPBR and PAB in four QTL Stg and the recurrent parent (RTx7000) grown under conditions with water limitation at two culture densities (LD = 10 plants / m2; HD = 20) plants / m2).

Figure 34 is a graphical representation showing the relationship between GLAA and PPBR in four QTL Stg and the recurrent parent (RTx7000) grown under conditions with water limitation at two culture densities (LD = 10 plants / m2; HD = 20) plants / m2).

Figure 35 is a graphical representation showing the relationship between GLAA and PASB in four QTL Stg and the progenitor recurrent (RTx7000) grown under conditions with limited water at two cultivation densities (LD = 10 plants / m2, HD = 20 plants / m2).

Figure 36 is a graphic representation showing the relationship between PPBR and PAB in four QTL Stg and the recurrent parent (RTx7000) grown under conditions with water limitation at two culture densities (LD = 10 plants / m2; HD = 20) plants / m2).

Figure 37 is a graphic representation showing the relationship between PPBR and PASM in four QTL Stg and the recurrent parent (RTx7000) grown under conditions with water limitation at two culture densities (LD - 10 plants / m2; HD = 20 plants / m2).

Figure 38 is a graphic representation showing the relationship between PPBR and grain production in four QTL Stg and the recurrent parent (RTx7000) grown under conditions with water limitation at two cultivation densities (LD = 10 plants / m2; HD = 20 plants / m2).

Figure 39 is a graphical representation showing the relationship between RWC in the medium grain filling (FL-2) and the relative senescence range in four QTL Stg and the recurrent parent (RTx7000) grown under conditions with water limitation in two cultivation densities (LD = 10 plants / m2, HD = 20 plants / m2).

Figure 40 is a graphic representation showing the relationship between the relative senescence range of the leaf and the green leaf area at maturity in four QTL Stg and the recurrent parent (RTx7000) grown under conditions with limited water at two cultivation densities (LD = 10 plants / m2) HD = 20 plants / m2).

Figure 41 is a graphical representation showing the relationship between the relative water content (RWC) in a medium grain fill (FL-2), and the mass of the stem at maturity in four QTL Stg and the recurrent parent ( RTx7000) grown under conditions with water limitation and two cultivation densities (LD = 10 plants / m2, HD = 20 plants / m2).

Figure 42 is a graphical representation showing the relationship between the post-flowering stem mass (PASM) and the post-flowering biomass (PAB) in four QTL Stg and the recurrent parent (RTx7000) grown under constrained conditions of water in two cultivation densities (LD = 10 plants / m2, HD = 20 plants / m2).

Figure 43 is a graphical representation showing the relationship between post-flowering stem biomass and grain production in four QTL Stg and the parent of origin (RTx7000) grown under conditions with water limitation at two cultivation densities (LD = 10 plants / m2, HD = 20 plants / m2).

Figure 44 is a graphic representation showing the relationship between the posterior stem mass in flowering (PASM) and the subsequent biomass in flowering (PAB) in four QTL Stg1 and the recurrent parent (RTx7000) grown under conditions with limited water and two growing densities grown in an experiment in 2005 (LD = 10 plants / m2, HD = 20 plants / m2).

Figure 45 is a graphical representation showing the relationship between the post-flowering stem mass (PASM) and grain production in four QTL Stg and the recurrent parent (RTx7000) grown under conditions with water limitation at two densities of culture (LD = 10 plants / m2, HD = 20 plants / m2) grown in an experiment in 2005.

Figure 46 is a graphical representation showing the relationship between post-flowering stem mass (PASM) and post-flowering biomass (PAB) in various fine mapping lines Stg 1 and the recurrent parent (RTx7000) grown under conditions with water limitation in two cultivation densities (LD = 10 plants / m2, HD = 20 plants / m2).

Figure 47 is a graphical representation showing the relationship between the relative water content (RWC) in a medium grain fill (FL-2) and the grain production in various combinations of QTL Stg and the recurrent parent (RTx7000) grown under conditions with limited water at two cultivation densities (LD = 10 plants / m2, HD = 20 plants / m2) in an experiment grown in 2004.

Figure 48 is a graphical representation showing the relationship between the leaf water potential (LWP) of FL-2 in a medium grain filling (bars) and grain production (g / m2) in the QTL Stg1 ( 6078-1) and the recurrent parent (RTx7000) grown under conditions with limited water at two cultivation densities (LD = 10 plants / m2, HD - 20 plants / m2).

Figure 49 is a graphical representation showing the relationship between PPBR and CWU during the filling of grain in four QTL Stg and the recurrent parent (RTx7000) grown under conditions with limited water at two cultivation densities (LD = 10 plants / m2, HD = 20 plants / m2).

Figure 50 is a graphical representation showing the relationship between CWU during grain filling (mm) and grain production (g / m2) in four QTL Stg and the recurrent parent (RTx7000) grown under conditions with water limitation in two cultivation densities (LD = 10 plants / m2, HD = 20 plants / m2).

Figure 51 is a graphical representation showing the relationship between PPBR and grain production in four QTL Stg and the recurrent parent (RTx7000) grown under conditions with limited water at two cultivation densities (LD = 10 plants / m2; HD = 20 plants / m2).

Figure 52 is a graphical representation showing the relationship between CWU during grain filling (mm) and the grain size (mg) in four QTL Stg and the recurrent parent (RTx7000) grown under conditions of water limitation in two cultivation densities (LD = 10 plants / m2, HD = 20. plants / m2).

Figure 53 is a graphic representation showing the relationship between PPBR and grain size in four QTL Stg and the recurrent parent (RTx7000) grown under conditions with water limitation at two cultivation densities (LD = 10 plants / m2; HD = 20 plants / m2).

Figure 54 is a graphical representation showing the relationship between PPBR and CWU during grain filling in several fine mapping lines Stg1 and the recurrent parent (RTx7000) grown under conditions with limited water at two culture densities (LD = 10 plants / m2, HD = 20 plants / m2).

Figure 55 is a graphical representation showing the relationship between CWU during grain filling (mm) and grain production (g / m2) in several fine mapping lines Stg1 and the recurrent parent (RTx7000) grown under conditions with limitation of water in two cultivation densities (LD = 10 plants / m2, HD = 20 plants / m2).

Figures 56A to 56C are graphical representations showing the results of running a simulation model of sorghum culture using the Buster genetic variety with the usual 2 stems / plant (HT) versus a Buster variety only with 1 stem / plants (LT) in a well wet (WW) and terminally stressed (TS) virtual environment. For both virtual environments, the following parameters were chosen: plant density 5 plants / m2 with a row spacing of 1 m; ground depth = 1800 mm; PA ground WC = 324 mm; N not limiting.

Figure 57A is a graphic representation of the expression differential of SbPIN4 (candidate Stg1) under irrigation conditions. Under irrigation conditions, this gene is inhibited in young root tips NIL Tx642 and Stg1 in comparison with Tx7000.

Figure 57B is a graphical representation of the differential expression of SbPIN4 (candidate Stg1) under water deficient conditions. Under water-deficient conditions, this gene is increased in most tissues, but especially in the expansion leaves of NIL Tx642 and Stg1, compared to Tx7000.

Figure 57C is a graphic representation of the differential expression of SbPTN2 (candidate Stg2) under irrigation conditions. Under irrigation conditions, this gene is slightly increased in the tissues of the stem and root of NIL Tx642 and Stg1 compared to Tx7000.

Fig. 57D is a graphical representation of a differential expression of SbPIN2 (candidate Stg2) under conditions with water deficiency. Under conditions with Water deficiency, this gene is increased in most tissues of NIL Tx642 and Stg1 compared to Tx7000.

Detailed description of the invention Throughout the present specification, unless the context requires otherwise, the word "comprises", or variations such as "comprising" or "comprising", will be understood to imply the inclusion of an element or integer or step of method or group of elements or integer or steps of manifested methods, but does not imply the exclusion of any other element or integer or step of method or group of elements or integers or steps of methods.

As used in the present specification, the singular forms "a", "an", "an", and "the" include plural aspects unless the context clearly indicates otherwise. Therefore, for example, the reference to "one locus" includes a single locus, as well as two or more loci; with reference to "an auxin", a single auxin is included, as well as two or more auxins; the reference to "the description" includes only one aspect or multiple aspects taught by the description. The aspects taught here are comprised by the term "invention". All aspects taught, described or claimed in the present invention are allowed within the scope of the present disclosure.

The present disclosure teaches QTLs associated with, and which facilitate the ever green phenotype in crops that They include cereal plants. The QTLs are generically referred to as StgX where X is a number of 1 and greater, which corresponds to a genetic locus or region of genetic loci in a particular chromosome in a crop plant. A sub-region is referred to as StgXm, where m is an alphabetic designation of a region within StgX. The modulation of the expression of a StgX in all the tissue or tissue selected in a plant is taught in the present invention to facilitate a physiological and genetic network that induces or promotes a change of water by the crop plant in the period after flowering or water access capacity increased during crop growth or increased transpiration efficiency, to thereby increase the harvest index (Hl) and the grain production under conditions with water limitation. The "expression" of a StgX includes the increase or inhibition of expression levels (eg, expression modulation) of a locus, as well as the selection of a polymorphic variant that is expressed at a higher or lower level, or that encodes a more or less active product in all the tissue or tissue selected in a plant. The locus, itself, can confer this phenotype or a functional equivalent thereof, such as a cDNA that encodes the same protein encoded by the locus.

QTLs identify loci that encode PIN proteins. PIN proteins are transporters of auxin efflux that they contain transmembrane domains, and are located mainly in plasma membranes. The term "PIN" is derived from "pin-type inflorescence". A pin for sorghum is "SbPIN". The present description teaches a PIN of any plant (for example, OsPIN of rice). The genomic location of a Sorb SbPIN is described in Table 1A.

In the present invention, a method is provided for generating a genetically modified plant that uses water more efficiently than a non-genetically modified plant of the same species, wherein the method comprises introducing into a plant or progenitor of the plant a genetic agent that encodes a PIN protein or a functional homolog or ortholog thereof thereof; or that modulates the expression of a native PIN protein; where the level and location of the PIN expression facilitate an 'always green' phenotype, where the phenotype includes, among other things, a cover architecture that facilitates a change in the use of water in the post-flowering period or capacity increased access to water during crop growth, resulting in an increased rate of harvest and grain production under conditions with limited water.

The present disclosure also teaches a method for generating a genetically modified plant that uses water more efficiently than a non-genetically modified plant of the same species, wherein the method comprises introducing into a plant or progenitor of the plant, a genetic agent that encodes a sorbent SbPIN protein selected from SbPIN 1 to SbPIN 11 or an equivalent in another plant or that modulates the expression of a native PIN. Examples of PINs include SbPIN4 and SbPIN2 and other SbPINs described in Table 1A and their equivalents in another plant, as well as a PIN having a particularly desired polymorphic variation, which, for example, allows an altered expression profile of the levels Elevated protein PIN. Examples of PINs in other OsPIN5 plants, corresponding to SbPIN4 and OsPIN3a that correspond to SbPIN2.

The genetic agent can be a locus or genomic region or its functional equivalent, such as cDNA or genomic DNA fragment. Alternatively, the agent can modulate expression as a native PIN locus in a particular plant. By the term "introduction" is meant by recombinant intervention, by mutagenesis or seeding after selection.

Without intending to limit the technology to the present specification, in its theory or mode of action, the expression of modulation of a PIN alone or in combination with a genetic or physiological network, alters the architecture of the plant to increase or otherwise promote the efficient water use. In one aspect, the modified architecture is the architecture of the modified floor covering.

The term "progeny" includes an immediate progeny, as well as distant relative ones of the plant, provided that it expresses in stable form the trait 'always green' introduced first in a previous progenitor.

The reference to a "crop plant" includes a cereal plant. The cereal plants grown here, include sorghum, wheat, oats, corn, barley, rye, rice, abaca, alfalfa, almond, apple, asparagus, banana, beans, blackberry, beans, cañola, cashew, cassava, chickpea, citrus, coconut, coffee, corn, cotton, fig, flaxseed, grapes, peanut, hemp, kenaf, lavender, hand, fungus, olive, onion, pea, peanut, pear, millet, potato, ramie, rape seed, ryegras, soybean, strawberry, beet, cane sugar, sunflower, sweet potato, taro, tea, tobacco, tomato, triticale, truffle and yam. In one example, sorghum wilt tolerance mechanisms are used to promote tolerance to wilt in sorghum, as well as other crop plants. In one example, genetically modified plants use water more efficiently than a non-genetically modified plant of the same species. The existing PIN loci in each of the above plants are referred to as "native" PINs. The present disclosure teaches how to increase and inhibit a native PIN or select a PIN that has a particular expression profile. A "native" PIN is a PIN locus in a progenitor plant before any manipulation (recombinant, mutagenesis or of seeded).

The term "tolerance to wilting" includes wilt evasion, adaptation to wilting, resistance to wilting, reduced sensitivity to wilting conditions, increased water use efficiency, as well as the ability to change the use of water in the period after flowering or increased water access capacity during crop growth, to thereby increase Hl and grain production under conditions with water limitation. Plants that exhibit tolerance to wilting are described as "plants adapted to wilt" or "plants that exhibit reduced sensitivity to conditions with limited water". In the present invention it is taught that tolerance to wilt is induced, facilitated, or otherwise associated with the 'always green' phenotype.

By the term "genetically modified", in relation to a plant, a genetically modified plant originally derived is included, as well as any progeny, immediate or distant that stably expresses the 'always green' trait. Therefore, the present disclosure teaches both classical planting techniques for introducing the genetic agent, i.e. the 'always green' QTL or a functional equivalent thereof, such as cDNA or a genomic fragment or an agent that increases or inhibits (ie, module) the expression of QTL or the protein encoded by it, as well as genetic engineering technology. The latter is comprised of the terms "genetic engineering means" and "recombinant means." The markers that define the 'always green' mechanism can also be classified during planting protocols to monitor the transfer of particular genetic regions. In addition, a specific 'always green' region is genetically inserted through recombinant media without a plant cell or plant callus and a regenerated seedling. A "genetically modified" plant includes a progenitor or any progeny, as well as any plant products, such as grain, seed propagation material, pollen, and ovules. In addition, a PIN locus can be expressed in a particular plant tissue, but not expressed, or its expression reduced in another tissue. In addition, a plant can be subject to mutagenesis, such as radioactive or chemical mutagenesis, and selected mutated plants with a PIN having a desired phenotype.

Referring to the "ever green phenotype", selected characteristics of the architectural plasticity of the increased cover, reduced cover size, increased biomass per unit leaf area in flowering, higher transpiration efficiency, increased water use are included. during the filling of the grain, state of water in the plant increased during the filling of the grain, proportion reduced biomass pre: post flowering, delayed senescence, increased grain production, larger grain size and reduced fixation.

In the present invention, a method for generating a genetically modified plant that uses water more efficiently than a non-genetically modified plant of the same species is enabled, wherein the method comprises introducing into the plant or a progenitor of the plant, an agent genetic that encodes a product that is associated with, or that facilitates an ever green phenotype, wherein the phenotype includes a change in the use of water in the post-flowering period, or an increased water accessibility during the growth of the crop or a increased transpiration efficiency which results in an increased grain yield and yield index under conditions with water limitation, and wherein the product is selected from the line consisting of SbPINI to 11 including SbPIN4 and SbPIN2, and other SbPINs described in Table 1A or an equivalent thereof in another plant, or the agent that modulates the expression of a native PIN in the pl anta.

Therefore, the use of genetic material encoding a genetic material that modulates the levels of a native PIN to facilitate the 'always green' PIN phenotype is taught in the present invention.

In the present invention, plants are provided genetically modified plants that exhibit the 'always green' phenotype as a result of genetic modification, as well as seeds, fruits, flowers and other propagating or propagating material. Root stocks and propagation stocks are also enabled. This is based on the premise that the seeds, fruits, flowers, reproductive and propagating material, exhibit or can pass in the 'evergreen' introduced in the final parent (s).

Referring to an "agent that modulates the expression levels of a PIN", include promoters, microRNAs, genes and chemical compounds that facilitate the increased or decreased expression of the selected tissue or tissue gene or an increased or decreased activity of a product of gene, as well as cDNA and genomic fragments. An agent can also be an intron of a genomic gene that is part of a natural genetic network to facilitate the modulation of expression.

A PIN protein produces an auxin gradient in cells that contain a transmembrane domain and is located primarily on the plasma membrane. PIN proteins are the limiting factors of the auxin transport range and provide vector direction for auxin to flow. In the present invention it is taught that at least one of Stg1 or Stg2 encodes a PIN protein. The introduction of Stg1 or Stg2 de novo into a plant or the elevation of its modulation or expression of a native Stg1 or Stg2, to facilitate the display of one or more characteristics or sub-characteristics associated with the ever-green phenotype.

As indicated above, PIN proteins are auxin efflux transporters that transmit the transport of polar auxin (PAT) from cell to cell, as opposed to the transport of auxin through the xylem (Rashotte et al. : 1683-1697, 2000; Friml et al, Current Opinion in Plant Biology 6: 7-12, 2003). The term 'PIN' is derived from the pin-like inflorescence that develops in Arabidopsis, when transport is defective. In the present invention, a SbPINn of sorghum is enabled, where n is a number from 1 to 11, as well as a PIN of any other plant.

Also taught in the present invention, is a method for generating a genetically modified plant that uses water more efficiently than a non-genetically modified plant of the same species, wherein the method comprises introducing into a plant or progenitor of the plant, a genetic agent encoding a protein selected from the list consisting of SbPINI to 11 such as SbPIN4 and SbPIN2 or other SbPINs described in Table 1 A, or an equivalent thereof in another plant or an agent that modulates the expression of a PIN native on the plant.

In the present description it is taught that Sorb SbPIN4 and SbPIN2 are genes for adaptation to wilting important along with other SbPINs, as well as their equivalents in other plants. The differences in auxin signaling explain all the multiple phenotypes observed in plants with a PIN expression profile. The phenotypes exhibited by plants SbPIN4 or SbPIN2 are explained, for example, by changes in auxin efflux and include reduced shoot generation, smaller leaves (both in length and width), reduced leaf mass and a higher proportion increased at the root: outbreak. The phenotypes exhibited by plants SbPIN4 or SbPIN2 can also be explained, for example indirectly (as emergent consequences of these direct effects) and include the increased availability of water in flowering, higher N concentration in the leaf at flowering, perspiration increment and biomass per unit leaf area, higher transpiration efficiency, retention of the green area of the leaf during grain filling, increased harvest index, higher grain production, larger grain size and increased fixing resistance. In the present invention, equivalents of SbPIN4 and SbPIN2 and other SbPINs are provided through other important crop and cereal species, to increase the adaptation to wilting in regions worldwide, where water limits the growth of the subsequent crop to flowering.

In accordance with the teachings of this specification, the modulation of the expression of a PIN selected from SbPINI to 11, such as SbPIN4 (Stg1) and SbPIN2 (Stg2) or its equivalents in other plants throughout the tissue, or in selected tissues, confers adaptation to wilt both directly , as indirectly, leading eventually to greater grain production, larger grain size and resistance to fixation under water limiting conditions.

In one example, the 'always green' phenotype involves the presence of multiple proteins such as two or more SbPINa to 11, such as SbPIN4 and SbPIN2.

In the present invention a method is taught to generate a genetically modified plant that uses water more efficiently than a non-genetically modified plant of the same species, wherein the method comprises introducing into a plant or parent of the plant a genetic agent that encodes two or more PINs or a functional counterpart or orthologous thereof, which are associated with or facilitate an 'always green' phenotype, wherein the phenotype includes a change in the use of water in the post-flowering period, or a increased water accessibility during crop growth or increased transpiration efficiency which results in an increased harvest rate and grain production under conditions with water limitation; or an agent that modulates the levels of expression of two or more PINs.

By the term "two or more", it means 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11. A single or multiple PIN loci can also be expressed or inhibited.

In one example, the genetic material is selected from (i) an agent that encodes SbPIN4; and (ii) an agent that modulates the expression levels of SbPIN4 or its equivalent in another plant. In another example, the genetic material selected from (i) an agent encoding SbPIN2; and (ii) an agent that modulates the expression of SbPIN2 or its equivalent in another plant.

The availability of increased water in flowering is achieved by using reduced water due to two mechanisms (generation of reduced shoots and smaller leaves) in plants that contain the Stg1 region. Both mechanisms, individually, seem to reduce the size of the cover by approximately 9%, on average. The mechanism of "low stem generation" dominates in low density environments, when the generation potential of scions is high. The "small leaf" mechanism dominates in high density environments, where the potential of stem generation is low. In combination, these two mechanisms provide crop plants with considerable plasticity to modify the roof architecture in response to the severity of water limitation.

The 'always green' phenotype increases the plasticity of roof architecture through constitutive responses and adaptation. The cover size in Stg1 or Stg2 is reduced by approximately 5%, even when there is no water limitation (constitutive response). The cover size is further reduced (adaptation response) in moderate wilt (-10%) and more severe wilt (~ 15%). The low generation of offspring is mainly a constitutive response. The small leaf size is both a constitutive response and an adaptation one.

Furthermore, in the present invention it is taught that the region Stg1 or Stg2 confers adaptation to wilting, reducing cover size (by generating reduced scion and smaller leaves) and reducing water use in the crop at flowering. This is shown through a high correlation (r 2 = 0.9) between the size of the cover and the use of crop water in studies of artificial wilt (rain shelter [ROS]) and lysis meter.

Increased water availability was also achieved in flowering, through increased water access capacity (better water extraction) and deeper or larger lateral dispersion).

The 'always green' genotype increases the biomass per unit leaf area at flowering. Assuming that the mass of the root is equivalent (or at least not significantly smaller), these differences can be explained by differences in transpiration (T) per unit leaf area [LA] (T / LA) and / or transpiration efficiency (TE). Lysis measurement studies indicate that the increase in T / LA, instead of TE, leads to increases observed in biomass per leaf area. It should be noted that the increased T / LA only occurred when the water deficit was sufficient to reduce the area of the leaf. When the water deficit was less severe (ie not sufficient to reduce the area of the leaf) then T / LA decreased, resulting in a higher TE.

The upper TE in StgX lines, such as Stg1 lines or Stg2, was also observed when the water deficit is less severe. The TE increased by introgression of Stg1 or Stg2, is proposed as due to a) photosynthetic capacity proportionally greater in comparison with the stomatal conductance, due to smaller, thinner and greener leaves, and / or b) a decrease in perspiration keeping the same time the biomass. Lysimetry studies indicate that both of these mechanisms contribute to a higher TE in Stg1 lines, with the reduction in transpiration of the primary mechanism.

Changes in transpiration per unit leaf area are shown in the present invention, as due to a) number of stomata, b) stomatal opening size, c) changes in opening and closing synchronization of stomata relative to VPD, and / od) the number of hair-based cells (which affect the layer of the limit and therefore T / LA). For example, the introgression of Stg 1, for example, in RTx7000, modified the anatomy of the leaf by increasing the number of packet wrap cells surrounding the vascular package.

The differences in leaf morphology can be seen between RTx7000 and Stg1 or Stg2. In this case, there were more package wrappings, and smaller ones surrounding the vascular package in Stg1 or Stg2. Also, there were fewer stomata, and fewer ciliated cells per unitary leaf area (leaves 7 and 10) in Stg1 compared to RTx7000.

The use of increased water during grain filling is achieved through (i) increased water availability at flowering and (ii) increased water access capacity (better water extraction and deeper or greater lateral dispersion) during filling of the grain.

The use of culture water (CWU) before flowering was negatively correlated with CWU after flowering in an artificial wilt experiment (rain shelter [ROS]). For example, in one experiment, a 25% increase in the use of water after flowering (80 vs 60 mm) resulted in a 25% increase in grain production (400 vs 300 g / m2). This resulted in 50 kg / ha of grain for each additional mm of available water.

The use of increased water during the period of grain filling was exhibited by Stg1 or Stg2 under both treatments of low as high density in a storm protection experiment (ROS). This was mainly due to (i) use of reduced water in flowering under high density, and (ii) increased water accessibility during grain filling under low density.

In the present invention it is taught that StgX, such as Stg1 or Stg2 confer adaptation to wilting by association with pre- and post-flowering biomass production. The region Stg1 or Stg2, for example, reduces the proportion of biomass pre: post flowering below a critical level, increasing grain production and resistance to fixation.

In accordance with the teachings of the present disclosure, the expression of StgX, such as Stg1, Stg2, Stg3 and / or Stg4, facilitates one or more of the following phenotypes: (i) oldness of the delayed leaf (always green), greater production of the grain and resistance to the fixation are consequences of a greater state of water in the plant during the filling of the grain (due to the use of increased water during the filling of the grain ); (ii) the introgression of StgX, for example, in the RTx7000 background increases the state of water in the plant in moderate grain filling, as indicated by a) higher relative water content (RWC), and b) lower potential of water on the sheet (LWP): (Mi) greater grain production and larger grain size are consequences of increased water availability during grain filling; (iv) higher grain production, larger grain size and increased binding strength are not mutually exclusive (ie all three traits are exhibited by StgX); (v) the advantages of production and grain size are relatively greater under severe terminal wilting than under moderate terminal wilting; (vi) the benefit of the "always green" genes, for example in the RTx7000 background (innate) occurs in the production range of 1-3 t / ha (12 to 22%), followed by a minor benefit still significant in the performance range 3-4 t / ha (8 to 10%). However, there was a small penalty associated with these regions (2 to 4%) in higher production levels (5 to 8 t / ha) due to higher humidity conditions. It should be noted that these production ranges can be considerably higher in hybrids. Since the production of average sorghum grain for hybrids in the northern grain belt is about 2.5 t / ha, the benefit of the "ever green" genes should be significant. The reduction in grain production under conditions of higher humidity (without water limitation) due to the "always green" mechanism has not been observed in hybrids. (vii) the impact of StgX, for example, on RTx7000 it also increases grain size by 11%, on average, under severe terminal wilt. There was no impact of the StgX QTL on the size of the grain under a moderate terminal wilt or without wilt; Y (viii) each of the key StgX mechanisms is mapped to a defined region, suggesting that the action of a single gene has multiple pleiotropic effects.

The present description also teaches a business model to increase economic profits in grain production. In accordance with these teachings, a business model is provided for increased economic gains in crop production, wherein the model comprises generating crop plants having a PIN expression profile that results in the crop plant having a change in the use of water through the plant in the period after flowering, or an increased water access capacity during the growth of the crop, to thereby increase the Hl and the production of grain under conditions with limited water , obtaining seeds of the cultivated plant generated, and distributing the seeds to the producers of grain to obtain a production and increased profits. With reference to PIN, SbPINI is included to 11 such as SbPIN4 and SbPIN2, as well as their equivalents in other plants.

In the present invention, a management system is taught of plants to reduce the dependence of crops on water, or to improve otherwise the efficiency of water use and to increase the production of grains or products. The plant management system includes the generation of a culture adapted to wilt, including cereal plants, using the selection and modulated expression of the PIN locus or its functional equivalent, as defined in the present invention alone or in combination with the introduction of other useful features such as grain size, root size, salt tolerance, herbicide resistance, pest resistance and the like. Alternatively or additionally, the plant management system comprises the generation of plants adapted to wilting and agricultural procedures such as irrigation, nutrient requirement, density and crop geometry, weed control, insect control, aerated soil, reduced shoot generation, raised beds and the like.

The present specification is an instruction as a means to induce or increase the ability to adapt to wilting in a plant, introducing de novo one or more characteristics of the "always green" phenotype, or raising or reducing the expression of one or more PIN loci. existing in a plant and / or by selecting a polymorphic PIN variant with increased or improved product expression or activity. The manipulation of the "always green" phenotype can be carried out out alone or as part of an integrated plant management system, which may include a trait selection and / or additional improved agronomic techniques. The resulting crops use water more efficiently, and have higher grain production and increased grain size.

The teachings of the present invention include business models for collecting seeds from crop plants increased or adapted to wilt for distribution to producers to ultimately increase grain production.

The present disclosure is enabling with respect to the use of a genetic agent that encodes a PIN protein, or that modulates the expression levels of a native PIN protein in the manufacture of a plant adapted to wilt.

The QTLs are identified in the present invention as carrying one or more PIN loci, where the level of expression is selected in seeding protocols or by genetic engineering, to promote the "always green" phenotype.

Genetically modified plants and their progeny, which exhibit the "ever green" trait, are also shown in the present invention, as well as seeds, fruits and flowers and other propagating or propagating materials.

The genetic material that encodes a PIN protein or a functional homologue or orthologous thereof, which is associated with, or facilitates an ever green phenotype, where the phenotype includes a cover architecture that facilitates a change in the use of water in the flowering period or an increased water accessibility during crop growth or increased transpiration efficiency , results in an increased harvest and grain yield index under conditions with water limitation; and an agent that modulates the PIN level; both being shown in the present specification.

In one example, the genetic material is selected from (i) an agent encoding an SbPIN described in Table 1A; and (ii) an agent that modulates the expression levels of a SbPIN described in Table 1A or its equivalent in another plant.

In one example, the genetic material is selected from (i) an agent that encodes SbPIN4; and (ii) an agent that modulates the expression levels of SbPIN4 or its equivalent in another plant. In another example, the genetic material selected from (i) an agent encoding SbPIN2; and (ii) an agent that modulates the expression of SbPIN2 or its equivalent in another plant.

The genetic material includes an agent that encodes a PIN or homologous or functional ortholog of the same, which is associated with, or facilitates an "always green" phenotype, where the phenotype includes a cover architecture that facilitates a change in the use of water in the period after flowering, or an increased water access capacity during the crop growth or increased transpiration efficiency resulting in an increased crop yield and grain yield under conditions with water limitation. The active agent includes an agent that increases or inhibits PIN levels. The genes in question can also be used as markers to transfer regions of genomes that comprise one or more of the genes, or their equivalents in other plants, to a particular plant, in order to include the "always green" phenotype.

EXAMPLES The aspects shown and enabled in the present invention are described further through the following non-limiting examples.

EXAMPLE 1 Identification of a StgX gene A quantitative trait locus (QTL) has been identified, which is referred to in the present invention as Stg1, which is an example of a StgX, which increases or improves the efficiency of water use in sorghum plants. Stg1 encodes a bicolor member of sorghum from the family of component 4 auxin efflux transporter, PIN4 (or SbPIN4).

This adaptation gene to the important wilt has been mapped in fine form in the sorghum genome. Changes in auxin efflux could explain all the multiple phenotypes observed in plants containing SbPIN4. The Gen The candidate (and promoter region) is sequenced in both parents of the fine mapping population (RTx7000 and Tx642) to identify a single nucleotide polymorphism. The profiling of the RNA expression of the population of fine mapping Stg1, is also carried out in a subset of lines, times and organs. The phenotypes exhibited by SbPIN4 plants that can be directly explained by the changes in auxin efflux, include reduced shoot generation, smaller leaves (both length and width), reduced leaf mass and increased root / leaf ratio. The phenotypes exhibited by SbPIN4 plants that can be explained indirectly (or as consequences that arise from these direct effects), include increased water availability at flowering, higher N concentration in the leaf at flowering, transpiration and biomass per area of unitary leaf increases, reduced proportion of biomass pre: post flowering, greater efficiency of transpiration, retention of green leaf area during grain filling, increased harvest index, higher grain production, larger grain size and resistance to fixation increased. It is proposed that SbPIN4 operates through other important cereal and crop species to increase wilt adaptation in regions worldwide, where water limits post-flowering crop growth.

Stg1 (SbPIN4) confers adaptation to wilting both directly, as indirectly, eventually leading to greater grain production, larger grain size and resistance to fixation under conditions with limited water.

The availability of increased water in flowering is achieved through the reduced use of water due to two mechanisms (generation of reduced rods and smaller leaves) in plants that contain the Stg1 region. Both mechanisms, individually, seem to reduce the size of the cover by approximately 91%, on average. The mechanism of "low stem generation" dominates in low density environments, when the generation potential of scions is high. The "small leaf" mechanism dominates in high density environments where the potential for rod generation is low. Combined, these two mechanisms provide crop plants with considerable plasticity to modify the architecture of the cover, in response to the severity of the water limitation.

The "always green" feature increases the plasticity of the roof architecture through constitutive and adaptive responses. The size of the cover in Stg1 is reduced by approximately 5%, even when water is not limited (constitutive response). The size of the cover is further reduced (adaptation response) in moderate wilt (-10%) and more severe wilt (-15%). The low Stem generation is primarily a constitutive response. Small leaf size is both a constitutive and adaptive response.

There is a link between reduced cover size (through the generation of reduced stems and smaller leaves) and the use of water in reduced culture at flowering. The high correlation (r2 = 0.9) between the size of the cover and the use of crop water in ROS studies and lysis measurement.

Water availability is also increased in flowering, through the increased access to water (better water extraction and deeper or larger lateral dispersion).

The always green trait increases the biomass per unit leaf area at flowering. Assuming that the mass of the root is equivalent (or at least not significantly smaller), these differences can be explained by the differences in transpiration per unit leaf area (T / LA) and / or transpiration efficiency (TE). Lysis measurement studies indicate that increases in T / LA, instead of TE, lead to the observed increases in biomass per leaf area. It should be noted that the increased T / LA occurred only under low VPD conditions; T / LA was actually reduced under high VPD conditions, presumably as a water conservation mechanism.

Higher TE was also observed in Stg1 lines, under higher VPD conditions. The TE increased by introgression of Stg1, may be due to a) proportionally greater photosynthetic capacity in comparison with the stomatal conductance, due to smaller, thinner and greener leaves and / or b) a decrease in perspiration, while maintaining the biomass Lysis measurement studies indicate that both of these mechanisms contribute to higher TE in Stg1 lines, with the reduction in transpiration of the primary mechanism.

Changes in transpiration per unit leaf area may be due to a) number of stomata, b) size of stomatal opening, c) change in opening timing and stomatal closure relative to VPD, and / or d) the number of cells of ciliated base (affecting the limit layer and therefore T / LA). The introgression of Stg1 in RTx7000, reduces the number of stomata and increases the number of hair-based cells per unit leaf area in leaves 7 and 10; both mechanisms can conserve water by reducing T / LA.

The introgression of Stg1 in RTx7000, modified the anatomy of the leaf increasing the number of packet wrapping cells that surround the vascular package. The increased number of cells in the package wrapper can also contribute to increased photosynthetic assimilation and therefore TE.

The differences in the morphology of the leaves (for example, leaves 7 and 10) can be seen between Tx7000 and Stg1. In this case, there were more package and smaller envelopes surrounding the vascular package in Stg1. The increased number of cells in the package envelope must also contribute to increased photosynthetic assimilation and therefore TE.

The use of increased water during grain filling is achieved through (i) increased water availability at flowering, and (ii) increased water access capacity (better water extraction and deeper or greater lateral dispersion) during filling of the grain. a) Availability of increased water in flowering The use of culture water (CWU) before flowering was negatively correlated with CWU after flowering in a ROS experiment. For example, in one experiment, a 25% increase in the use of water after flowering (80 vs 60 mm) resulted in a 25% increase in grain production (400 vs 300 g / m2). This resulted in 50 kg / ha of grain for each additional mm of available water. b) Increased water access capacity during grain filling The use of increased water during the period of grain filling was exhibited by Stg1 under treatments with both low and high density in a ROS experiment. This was mainly due to (i) use of reduced water at low flowering high density, and (ii) increased water access capacity during grain filling under low density.

The Stg1 region confers adaptation to wilting through a union between pre- and post-flowering biomass production. The Stg 1 region reduces the proportion of biomass pre: post flowering below a critical level, increasing grain production and resistance to fixation.

The aging of the delayed leaf ("always green"), greater grain yield and resistance to fixation, are consequences of a state of water in the upper plant during the filling of the grain (due to the use of increased water during the filling of the grain).

The introgression of Stg1 into an RTx7000 background increased the water status of the plant in moderate grain filling, as indicated by a) relatively higher water content (RWC), and b) water potential of the smaller leaf ( LWP).

Higher grain yield and larger grain size are consequences of increased water availability during grain filling.

Greater grain yield, larger grain size and increased binding strength are not mutually exclusive (ie all three traits are exhibited by Stg1).

The advantages of grain production and size are relatively greater under severe terminal wilting than low moderate terminal wilt.

Studies indicate that the greatest benefit of ever-green genes occurs in an RTx7000 (innate) background within the production range of 1 to 3 t / ha (12-22%), followed by a lower benefit, although still significant in the range of production 3 to 4 t / ha (8 to 10%). However, this was a small penalty associated with these regions (2 to 4%) in higher production levels (5 to 8 t / ha), due to water conditions. It should be noted that these production ranges can be considerably higher in hybrids. Since the average sorghum grain production for hybrids in the northern grain belt is about 2.5 t / ha, the benefit of the "evergreen" genes should be significant. In general, no reduction in grain production has been observed under conditions of higher humidity (no water limit) due to the always green trait.

The introgression of Stg1 in RTx7000, also increased grain size by 11% on average, under severe terminal wilt. There was no impact of this QTL on the size of the grain under a moderate terminal wilt or without wilt.

Each of the key Stg1 mechanisms maps to the same region, indicating the action of a single gene with multiple pleiotropic effects.

EXAMPLE 2 Reduced shoot generation (physiological studies of NlLs in the field) The data show the impact of Stg1 on the generation of shoots under conditions of both high-level water (HW) and low-level water (LW). The differences in the development of cover before flowering were largely a consequence of the variation in the generation of shoots between the lines. The number of rootlets per m2 in flowering was the best general measure of the effect of the generation of shoots in cover dynamics. The numbers of rootlets per m2 were equivalent under both water regimes (12.89), which indicates that the generation of reduced scion is a constitutive feature. The genotypes varied significantly (P <0.001) in this parameter, fluctuating from 8.59 to 16.67. However, the genotype and treatment did not interact significantly in this parameter.

The numbers of rootlets per m2 were analyzed in terms of their Stg states, and the category averages are presented in table 1. RTx7000 produced 41% more (P <0.05) rootlets / m2 than B35 (14.07 vs. 10.00). The introgression of the Stg1 region alone in RTx7000 (6078-1) reduced rootlets / m2 significantly (P <0.05) compared to RTx7000 (9.40 vs. 14.07). In comparison only with Stg1, additional introgressions of either Stg2 or Stg4 increased the numbers of rootlets at 10.49 (combination 1.2) and 10.74 (combination 1.4). It should be noted that the three quasi-isolines that do not contain Stg regions (2212-3, 2235-11 and 6120-16), also exhibited a high generation of offshoots equivalent to Tx7000. Therefore, the general fluctuation of rod generation in these lines is Stg1 < B35 < Stg4 < Stg2 < Stg3 < no < RTx7000.

At flowering, the numbers of rootlets were highly correlated (r2 = 0.71) with the total green leaf area (GLAA, figure 1).

Table 1 The rootlets per m2 in the recurrent parent lines (RTx7000) and in various lines containing QTL Stg1 introgressions, either alone or in combination with other QTL Stg.

The differences in the area of green leaf in flowering (GLAA), were mainly due to differences in the area of green leaf stem in flowering (GLAAt), since GLAAt was highly correlated with GLAA (r = 0.78), although the main leaf area was not.

The area of green leaf of stem in flowering was analyzed in term of state Stg and the averages of the category are presented in table 2. RTx7000 produced GLAAt almost eight times more (P <0.05) than B35 (15460 vs. 1980). The introgression of the Stg1 region alone in RTx7000 (6078-1) significantly reduced GLAAT (P <0.05) compared to RTx7000 (3121 vs 15460). In comparison only with Stg1, additional introgressions of either Stg2 or Stg4 increased GLAAT to 4187 (combination 1.2) and 4797 (combination 1.4). All lines containing Stg1 (in any combination) were not significantly different (P <0.05) in GLAAt from Stg1 alone. It should be noted that GLAAt in the three quasi-isolines that do not contain Stg regions (2212-3, 2235-11 and 6120-16) were not significantly different from RTx7000. Therefore, the significantly different classifications (P <0.05) of GLAAt in these lines are B35 = Stg1 = Stg4 < Stg2 = Stg3 < none = RTx7000.

Table 2 The area of green leaf stem at flowering in the recurrent parent lines (RTx7000) and in various lines contain QTL Stg1 introgressions, either alone or in combination with other QTL Stg.

The area of green leaf in rootlets per m2 at flowering was highly correlated under treatments of high (HD) and low (LD) density in the rain shelter experiment (figure 2). Introgression in the Stg1 or Stgla regions in RTx7000, reduced the rootlets per m2 and GLAA under both densities.

EXAMPLE 3 Reduced shoot generation (fine mapping studies) A population of fine mapping Stg1 was grown in the field under high and low density conditions in three years Consecutive The number of rootlets per plant was measured at flowering in each year, and a combined analysis was carried out over the years. In general, RTx7000 produced 47% more rootlets per plant than 6078-1 (1.85 vs. 1.26, Figure 3).

In these field studies, the trait was mapped (number of rootlets per plant). An arbitrary value of 1.54 rootlets per plant, produced the optimal separation between the generation of high and low level shoot for mapping purposes (for example, recombinants with less than 1.54 rootlets were BB genotypes, although those with more than 1.54 rootlets were genotypes TT). The decrease through the markers reveals that the gain of function was achieved (generation of low-level scions) in three genotypes (10564-2, 10704-1 and 10620-4). One recombinant (10568-2) exhibited a high-generation shoot phenotype, although three others (10620-4, 10704-1 and 10564-2) exhibited low-generation shoot phenotypes. An auxin 5 efflux transporter component gene was proposed as the candidate gene.

EXAMPLE 4 Reduced rod generation (fine mapping studies Stg1 in ROS) A subset of the population of fine mapping Stg 1 was grown in the field in the study of protection against rain (ROS) under high and low level water conditions, with each water treatment divided for high and low density. This created four water regimes with increasing levels of water deficit: HWLD (less stress) < HWHD < LWLD < LWHD (more tension). The number of rootlets per plant was measured 44 days after the emergence of each trace. The differences were the most obvious in the treatment of low density (LD), since the expression of stem generation is maximized in this treatment. On average, RTx7000, 10568-2 and 10709-5 produced 27% more rootlets per plant than 6078-1 and 10604-5 under LWLD conditions (2.05 vs. 1.62, Figure 4), and 23% more rootlets per plant under HWLD conditions (1.49 vs. 1.22).

EXAMPLE 5 Reduced rod generation (fine mapping studies Stg1 in igloo) Three additional fine mapping studies were conducted in the Stg1 population under controlled conditions in an igloo. In these studies, shoot generation was analyzed in more detail compared to earlier field studies. The total number of stem generators was counted and more specifically the number of stem generators that arose from the axilla of leaves 2 (T2), 3 (T3) and 4 (T4) was counted. The presence or absence of T2 was the best indicator of the general shoot generation potential of a given recombinant. T2 was also the best trait to be used for fine mapping of the gene.

In this experiment, the total number of generators stems was the sum of T2, T3 and T4, where T2 was the stem generator that emerges from the axilla of leaf 2 (and so on for T3 and T4), including secondary stem generators. Significant genotypic variation was observed in all the traits related to the generation of offspring in this study (Table 34), with inheritance capacities generally greater than 30.

Table 3 of Kindred tillerno_max presence_T2 presence_T3 presence_T4 gleaf_T stem_T bio_T b.o.GLA_T_ratio Table 3 provides a summary of the anticipated averages, the P value and the inheritance capacity of the shoot generation traits measured in harvest L11. The significant differences were shaded with yellow (P <0.05), while the inheritance capabilities > 20 were shaded with green.

The separate analysis of data T2, T3 and T4, found that 6078-1 did not produce T2 stem generators in any of the four replicas, while RTx7000 produced T2 stem generators in 2 out of 4 replicas (Figure 5).

There were also differences in the T3 numbers. 6078-1 produced a T3 stem generator in 1 of 4 replicates, while RTx7000 produced a T3 stem generator in all 4 replicates (Figure 6).

The numbers of stem generators T4 also varied among the genotypes. 6078-1 produced a T4 stem generator in 3 out of 4 replicas, while RTx7000 produced a T4 in all 4 replicas. Therefore, the introgression of Stg1 essentially prevented the growth of the T2 and T3 titulators in an RTx7000 background.

The lines that produced a T2 stem generator were separated into 0 to 2 replicas (low generation stem group, 8 recombinants) of those that produced a T2 stem generator in 3 to 4 replicas (high stem generation group, 8 recombinants ) [Table 4, figure 7].

Table 4 Table 4 shows the presence of stem generators (T1-T3) and the total number of stem generators of eight recombinants with high-level stem generation (brown shading) and eight low-level stem generation recombinants (shading green color) of the population of fine mapping Stg1.

Function gain (low level stem generation) is achieved in 10604-1-157-5 recombinant. Function gain (low generation of scion) is also achieved in three recombinants (10604-1-195-5, 10604-1-56-7, 10604-1-477-4).

A subset of lines was used in this experiment to validate the stem generation region. They were used more replicates per recombinant (20) to further reduce the error variance and increase the discrimination power between the lines. The results indicated the presence of an auxin efflux corner gene.

A breakpoint analysis of these lines was carried out, which, according to their genotype (BB or TT), "increased" or "decreased" through the region of interest, to identify the generation gene of low level stem. A clear break was seen, separating the lines that produced a total stem generator number of > 2.5 (high-level stem generation group; 5 recombinants) of those that produced a total stem generator number of < 2.5 (group of low-level stem generators, 3 recombinants) [Table 5, figure 8].

Table 5 Table 5 shows the presence of stem generators (T1-T4), including stem generators Secondary, and a total stem generator number for five high-level stem generation recombinants (brown shading) and three low-level stem generation recombinants (green shading) of the fine mapping population Stg1.

Function gain (low level stem generation) is achieved in 10604-1-157-5 recombinant. The PIN4 gene in bicolor Sorghum (here designated as "SbPIN4") is therefore a strong candidate for the low-generation rod gene Stg1.

EXAMPLE 6 Smaller leaves In general, the introgression of the Stg1 region in RTx7000, reduced the size of the leaf (length and width) under conditions of high irrigation and water limitation, indicating a constitutive gene action. However, the reduction in leaf size was generally greater under conditions with water limitation, indicating, to some extent, an adaptation response (inducible) in addition to the constitutive response. Therefore, Stg1 confers two mechanisms to reduce the cover size: a) reduced stem generation, and b) reduced blade size. Combined, these two mechanisms provide a high degree of plasticity for the plant to modify the architecture of the roof in response to environmental and / or management factors.

A series of lysis measurement studies particularly instruct in the evaluation of leaf size patterns under various levels of vapor pressure deficit (VPD) (Figures 9A to D). Although the pots were regularly irrigated in both experiments, the size of the cover differed between stations, presumably due to differences in temperature season and VPD, creating conditions of high (1.8 kPa) and low (1.3 kPa) VPD. For the main and the larger stem generator (T3), the reduction in leaf size was significant under both high and low VPD conditions (Figures 9A to D), although the reduction started later under a low VPD level compared to a high level VPD in the main stem (L12 vs L9) and T3 (L7 vs L5).

However, for the remaining rod generators (T4-T6), leaf size distributions differed markedly between experiments. Although there was no difference in leaf size between RTx7000 and 6078-1 under high-level VPD, the leaves of 6078-1 were significantly smaller under low-level VPD. This indicates an adaptation response (inducible) to the reduction of the sheet size under certain environmental conditions for the stem generators T4-T6.

Therefore, under low level water stress, the introgression of Stg1 in RTx7000 resulted in more sheets small on the main stem and larger stem generator (T3), but not on T4-T6. This response can enable the plant to maximize the interception of light in the rear stem generators, when conditions are favorable. Under high water stress, the introgression of Stg1 into RTx7000 resulted in smaller levels of all rootlets. This response may allow the plant to dramatically reduce its cover size under conditions with limited water.

EXAMPLE 7 Smaller leaves (Shelter studies Against Rain) Experiments on Rain Protection (ROS) were carried out to evaluate the impact of the Stg1 region under two cultivation densities, thus creating two levels of water deficit (high density = high voltage, low density = low voltage) . In general, shoot generation was low or absent under HD (20 plants / m2) and normal under LD (10 plants / m2).

The size of the cover was smaller during both years under the treatment of high density (HD), which reflects the greater deficit of water generated through this treatment. In both years under more moderate (LD) and more severe (HD) water deficits, leaf sizes were generally lower at 6078-1 (Stg1) compared to RTx7000. The exception was when the pattern of leaf size distribution was similar for 6078-1 and RTx7000 in the more moderate water deficit (LD), although the leaves were significantly smaller in 6078-1 (up to 18% smaller) under the greatest water deficit (HD), suggesting an adaptive response through the Stg1 plants to increase the water deficit. In fact, the introgression of the Stg1 region in RTx7000 reduced the size of the four largest leaves (L10-L13) by an average of 16.5% in the most severe water deficit (HD). Since there was little generation of offspring in any genotype in this treatment. The reduced leaf size in 6078-1 must have markedly decreased the size of the cover, and therefore the use of crop water (assuming similar transpiration per unit leaf area).

It should be noted that the mechanism of reducing the size of the leaf associated with Stg1 seemed to operate both in the presence (LD) and in the absence of stem generation (HD), although it seems to be better expressed under HD, where they usually occur uniraicilla conditions and high level water deficit.

EXAMPLE 8 Smaller leaves (study of fine mapping Stg1 in Shelter Against Rain) A subset of the population of fine mapping Stg1 was grown in the field in the study of Resguardo Contra Agua (ROS) under high and low level water conditions, with each water treatment dividing into high and low density. This created four water regimes with increasing levels of water deficit: HWLD (less stress) < HWHD < LWLD < LWHD (more tension). The area of each leaf of the fully expanded main stem was measured for all genotypes in all treatments.

The introgression of the entire Stg1 region (6078-1), and more specifically, the smaller region, designated 10604-5, resulted in a reduction in the size of leaves 9 to 13 under low and low water conditions. density (figure 10). For example compared to RTx7000, L11 was 9% and 16% smaller in 10604-5 and 6078-1, respectively. Since shoot generation was negligible in this treatment, differences in cover size were essenty due to differences in leaf size.

Under low density conditions, leaf size distributions were affected by stem generation, resulting in some crossings compared to high density treatment (see figure 10). However, it should be noted that 10604-5 produced smaller leaves 9 to 13 relative to 10709-5, 10568-2 and RTx7000 in both HD and LD treatments.

The "small leaf size" gene mapped for the gene candidate, the transporter component of auxin 5 efflux.

EXAMPLE 9 Smaller leaves (study of fine mapping in igloo) The area of the leaf varied significantly (P <0.001) among the genotypes with an inheritance capacity that reaches 60 for leaves 4 and 5. The introgression of the Sth1 region in RTx7000, reduced the area of leaves 1 to 6 ( figure 16). For example, the L6 area was 22% larger in RTx7000 (67.4 cm2) than 6078-1 (55.3 cm2), although this difference was not significant at the level. The area of L6 ranged from 47.8 cm2 to 93.9 cm2 (LSD [0.05] = 21), with an inheritance capacity of 42.

The differences in leaf area were due to more differences in the length of the leaf (figure 17) than in the width of the leaf (figure 13). Although the area of the leaf increased exponenty with the number of leaves (Figure 16), the length of the leaf increased in a linear fashion (Figure 12). The relation between the width and number of leaves was parabolic (figure 13). Therefore, the divergence in the area of the leaf between 6078-1 and RTx7000 with an increasing number of leaves, was mainly due to the divergence in leaf length between these genotypes. This suggests that the function of genes that reduce the size of the leaf is more likely associated with expansion (leaf length) than with cell division (leaf width).

The allometric relationship in the population of fine mapping Stg1 between the leaf area (n) and the leaf area (n + 1), indicates a significant change approximately on Sheet 8 (concurrently with the onset of flowering) ). Consequently, increases in leaf size occurred in a lower range. diomoaso -e rgi The introgression of the Stg1 region in RTx7000 reduced the area of leaves 9 to 11 [G) i VlPt | aenopoor- (figure 14), as well as leaves 1 to 6 (described above). The area of L9 varied significantly (P = 0.06) between the genotypes, fluctuating from 234 to 300 cm2, with d E Virerroaranaz an inheritance capacity of 21 (table 6). The L9 area was 8% larger in RTx7000 (263 cm2) than 6078SDE av-1 (244 cm2) [figure 14]. Similar trends can be seen for sheets 10 and 11.

Table 6 Sheet size L6_GLA 54,122 88,256 10,871 17,358 23,256 L7 GLA 103,157 0.317 213,237 14,083 14,156 16,947 L8_GLA 178,469 0.179 535.126 19.269 12.962 7.623 5.391 L9J3LA 266,844 673,230 18,676 9,724 45,684 L10 GLA 342,756 0.506 1029,389 9,361 1,177 0.455 L11_GLA 383,560 0.374 848,058 7,592 3,098 1,440 L11 DW 1,955 0.522 0.058 12,372 0.000 0.000 SLW L9 L11 50,083 L0.Q3O 17,372 3,073 8,322 0.956 18,041 Table 6 shows a summary of the anticipated averages, P value and inheritance capacity of the leaf size traits measured in crop L 11. Shaded with yellow significant differences (P <0.05), while the inheritance capabilities > 20 were shaded with green. GLA = green area of the leaf. DW = dry weight. SLW_L9_L11 = weight of the specific sheet.

Most of the variation in the area of green leaf in the harvest of Sheet 11, was due to differences in the generation of shoots. However, sheets 9 to 11 were smaller in 6078-1 compared to RTx7000. These differences were significant (P <0.05). It should be noted that the "generation of low-level offspring" and "small leaves" were both associated with the same region, indicating the possibility of a single gene that controls both features of the roof architecture.

EXAMPLE 10 Smaller leaves (studies of fine mapping in igloo) The number and length of the leaves were linearly correlated with the progenitors of the population of fine mapping Stg 1 (figure 15). The introgression of Stg1 into RTx 7000 resulted in a reduction in the length of leaves 8 to 10, with L10 being 7% shorter in 6078-1 than RTx7000 (550 vs. 592 mm).

For mapping purposes, the "tails" of the fine mapping population Stg1 were selected (figure 15). Two genotypes exhibited particularly long leaves (10604-1-157-5 and 10604-1-318-1) and three genotypes exhibited leaves particularly short (10604-1-222-1, 10604-1-501-327-3 and 6078-1).

Function gain (short sheet) was achieved in recombinant 10604-1-222-1. The "small leaf" gene is proposed to map the same region as the "low-level rod generation" gene.

For mapping purposes, the "tails" of the fine mapping population Stg1 were selected (figure 16). Three genotypes exhibited particularly long leaves (10604-1-157-5, 10604-1-318-1 and RTx7000) and two genotypes exhibited particularly short leaves (10604-1-222-1 and 6078-1).

By increasing the markers, the gain of function (short sheet) was achieved in recombinant 10604-1-222-1. Therefore, once again, the "small leaf" gene maps to the same region as the "low-level shoot generation" gene. It should be noted that the leaf length at L9 and L10, maps the same region.

One explanation is a simple gene with multiple pleiotropic effects. The increased availability of auxin may explain both the generation of low-level shoots and the small leaf-size phenotypes observed in plants that contain this region. A gene of auxin efflux transporter component 5 is located in the target region, and therefore is identified as a candidate.

EXAMPLE 11 The "always green" feature increases the plasticity of the roof architecture through answers constitutive and adaptation The availability of increased water in flowering is achieved through the use of reduced water due to two mechanisms (generation of reduced shoots and smaller leaves) in plants that contain the Stg1 region (figure 17). Both mechanisms, individually, seem to reduce the size of the cover by approximately 9% on average. The mechanism of "generation of low-level scions" dominates in low density environments when the generation potential of scions is high. The "small leaf" mechanism dominates in high density environments, when the potential of stem generation is low. Combined, these two mechanisms provide crop plants with considerable plasticity to modify the architecture of the canopy in response to the severity of water limitation.

The always green feature exhibits both constitutive and adaptive responses (figure 18). The size of the cover in Stg1 is reduced by approximately 5%, even when water is not limited (constitutive response). The size of the cover is reduced in an additional way (adaptation response) in a moderate wilt (-10%) and in more severe wilt (-15%). The generation of scion Low level is a primarily constitutive response, although the smaller leaf size in stem generators in response to increasing water deficit is an adaptive response. The smallest leaf size is a response that is both constitutive and adaptive.

EXAMPLE 12 Union between reduced cover size (through generation of reduced shoot and smaller leaves) and use of reduced culture water in flowering. The use of reduced culture water in flowering can be used through a) a smaller cover size with equivalent transpiration per unit leaf area, b) an equivalent cover size with less transpiration per unit leaf area, or c) a smaller cover size and less perspiration per unit leaf area. The ROS and lysis measurement studies indicate that under high water stress conditions, the Stg1 region, and in particular the recombinant containing the candidate gene Stg1 (10604-5), exhibited lower use of culture water due to a lower size of the roof, instead of less perspiration per unit leaf area. High correlations (r2 = 0.9) were observed between the cover size and the use of culture water, in ROS studies and lysis measurement. (a) Water savings due to smaller leaf size Stem generation was negligible in this experiment due to high cultivation density. Therefore, differences in cover size were due to differences in leaf size (Figure 20), as is clear from the high correlation between the size of the leaf 12 and the total green leaf area in the flowering (figure 21).

In turn, the area of green leaf at flowering was highly correlated with the use of water in the crop at flowering (figure 22). Relative to RTx7000, the two lines containing the candidate gene Stg1 (6078-1 and 10604-5), both exhibited smaller leaves (L10-L13), smaller green leaf area in flowering (GLAA) and lower use of crop water in bloom. (b) Water savings due to reduced transpiration per unit sheet area (asymmetry studies) The transpiration (T) is the product of the area of the leaf (LA) and the transpiration by area of the leaf (T / LA). Under conditions of high VPD, LA was similar between Stg1 and RTx7000 (11795 vs. 11628 cm2), although T / LA was lower in Stg1 than Tx7000 (2.60 vs. 2.85), resulting in lower water use per plant (T) in Stg1 than in RTx7000 (30.7 vs. 32.8 1). Therefore the water savings in Stg1 (in a high VPD environment) were completely achieved through a reduction in T / LA, which suggests that this is a constitutive water conservation strategy conferred by Stg1. In this case, the higher transpiration efficiency (TE) in Stg1, was a consequence of Equivalent biomass and low level transpiration.

A larger analysis compared to the four QTL Stg (Stg1, Stg2, Stg3 and Stg4) with RTx7000 helps put the Stg1 response in perspective. Under conditions of high VPD, T / LA was positively correlated with T. However, under conditions of low VPD, T / LA was negatively correlated with T (r2 = 0.52). The green leaf area and perspiration were correlated positively under both low and high VPD conditions (Figure 22). In both experiments, the QTLs reduced green leaf area and transpiration compared to RTx700G. (c) Water savings due to reduced leaf area The transpiration (T) is the product of LA and T / LA. Under conditions of low VPD, LA was 31% lower in Stg1 than RTx7000 (4898 vs. 7082 cm2). This was slightly offset by a 9% increase in T / LA in Stg 1 compared to RTx7000 (5.15 vs 4.70). The net result was a 22% reduction in the use of water per plant (T) in Stg1, compared to RTx7000 (25.6 vs 32.71), mainly due to the reduced cover size. The increase in T / LA exhibited by Stg1, can be by itself a mechanism of adaptation to wilting, by cooling the leaf and allowing photosynthesis to continue.

The plasticity in T / LA seems to be particularly important in the regulation of the water status of the plant.

Under conditions of high VPD, the reduced T / LA in Stg1 was the key mechanism to reduce T and increase TE. Under conditions of low VPD, T / LA increased in Stg1, could have contributed to the maintenance of leaf function by cooling.

Lysis measurement studies in a subset of fine mapping Stg1 provide additional insight into this region. Under conditions of high VPD, LA per plant was lower in 10604-5 (location of candidate gene Stg1) than RTx7000 (10283 vs. 1.1628 cm.2), although T / LA was equivalent in 10604-5 and RTx7000 (~ 2.86), resulting in less water use per plant at 10604-5 than RTx7000 (28.0 vs. 32.8 1). Therefore, the water savings in 10604-5 were completely achieved through a reduction in the size of the cover.

Under low VPD conditions, LA per plant was lower in all Stg1 lines compared to RTx7000, resulting in water savings in all Stg1 lines. Therefore, it was difficult to map this region in fine form, since all the recombinants responded in a similar way to 6078-1. (d) Simulation of Agronomy Macho planted in 5 plants / m2 with a row spacing of 1 m. Depth of earth = 1800 mm; PAWC of earth = 324mm; N not limiting. The results are shown in the Figures 56A to C.

Treatments HT Generation of high level shanks (2 shank / plant generators) LT Low level stem generator (1 stem / plant generator) WW Well watered (Starting with 100% profile and rain feeding) TS Terminal Tension (Beginning with profile in half [162mm] and without rain after establishment).

EXAMPLE 13 The availability of increased water in flowering can also be achieved through increased water access capacity due to better water extraction and / or deeper or greater lateral dispersion of roots in plants containing the Stg1 region. Root and root / shoot ratio (figure 23) were greater in Stg1 than RTx7000 in the stage of Leaf 6. There was a considerable transgression segregation for these traits in the population of fine mapping Stg1. The relationship between the root mass and the root: shoot ratio indicates the opportunity for additional genetic advancement in these traits.

The ratio of root mass per leaf area can be used as an index of wilting adaptation in the sowing stage, since it integrates the capacity of the plant to access to water (root mass) with the capacity of the plant to use the water (leaf area). A higher index indicates a greater capacity to access water per unit sheet area. Stg1 exhibited a greater proportion of root mass per leaf area relative to RTx7000, due to both a greater root mass and a smaller leaf area.

The greater proportion of root mass per leaf area exhibited by Stg1 in stage L6, may explain why it uses more water in the early growth of the crop (20 to 50 DAE) compared to RTx7000 under the LWLD treatment (figure 24). It is not clear yet whether the increased water access capacity during grain filling exhibited by Stg1 compared to RTx7000 (Figure 24) was due to better water extraction and / or higher root dispersion.

In a root camera experiment in Gatton, Queensland, Australia (Van Oosterom et al., 2010 supra), the interior gravimetric boundary of water extraction was 0.26% lower for A35 hybrids (always green features) than AQL39 (old age). A35 contains the region Stg1, while AQL39 does not. Assuming a volume requirement of 1.3 g cm-3 and a ground depth of 150 cm, this could potentially increase the water available in the field in > 5 mm throughout the life cycle of the crop.

EXAMPLE 14 The 'always green' trait increases the biomass per unit leaf area at flowering. Assuming that the root mass is equivalent (or at least not significantly smaller), these differences can be explained by differences in transpiration per unit sheet area and / or transpiration efficiency.

The biomass of the main stem per unit leaf area (B / LA) at flowering, was -24% higher in Stg1 than RTx7000 under low water tension conditions (35.2 vs 26.2 g / m2 / cm2) and high tension of water (40.6 vs 31.4 g / m2 / cm2) (Table 7). The B / LA of the main stem at flowering was ~ 14% higher under high water stress conditions than under low water stress conditions for both Stg1 and RTx7000, that is, B / LA increased with a water deficit . It should be noted that the B / LA of the stem generator was equivalent in Stg1 and RTx7000 in conditions of low voltage and high water voltage.

Table 7 Table 7 shows the biomass of the main stem, shoot generator and total per leaf area of RTx7000 (recurrent parent) and a number of almost isogenic lines containing several Stg1 introgressions grown in high and low water stress in Biloela, Queensland, Australia.

Detailed measurements of water use suggest that the highest biomass per unit leaf area observed in the Stg1 lines in Biloela was probably due to greater transpiration per unit leaf area instead of TE.

Low water tension Stg1 and RTx7000 showed B / LA equivalent in low water tension. However, T was ~ 7% lower in Stg1, due to the ~ 10% lower of T / LA, which, in turn, increased TE by ~ 9% (Figure 45). Therefore, Stg1 maintained the biomass, but used less water compared to RTx7000. High water tension At high water stress, B / LA was ~ 6% higher in Stg1 compared to RTx7000. B / LA correlated positively with T / LA but not with TE. Therefore, the higher B / LA shown by Stg1 was due to the higher T / LA. In general, B / LA correlated positively with T / LA and correlated negatively with TE in high water tension.

In this case, Stg1 used ~ 22% less water than RTx7000 during the period before flowering. Therefore, Stg1 can have significantly more water available for grain filling, despite the lower biomass at flowering.

EXAMPLE 15 The TE increased by the introgression of Stg1, may be due to a) photosynthetic capacity provides / greater in comparison with stomatal conductance, due to smaller, thinner and greener leaves, or b) a decrease in transpiration per area of leaf, while maintaining the biomass per leaf area In the population of fine mapping Stg1, the length and verdure (SPAD) of leaf 10, were correlated in negative (r2 = 0.72, figure 25). Therefore, the decrease in leaf size in this population increased the concentration of nitrogen in the leaf. The introgression of Stg1 into an RTx7000 background decreased the L10 length by ~ 7% (from 592 to 550 mm) and increased L10 SPAD by -4% (from 47.1 to 48.9).

The greener leaves can increase the photosynthetic capacity, and therefore, the efficiency of water use. In a subset of the population of fine mapping Stg1, photosynthesis increased with the SPAD value until it reached a plateau in a SPAD of ~ 48.5 (figure 26). However, line (6078-1) with the highest SPAD value (51.6) exhibited a relatively low range of photosynthesis (32.1 MJ / m / d). This result is either a) anomalous, or b) indicates a real decrease in photosynthesis at high SPAD values.

The greenness (SPAD) and WUE of the sheet (based on an index calculated by the Liquor software) were positively correlated in a subset of the fine mapping population Stg1 (Figure 27). None of the Stg1 introgressions reached the value of 6078-1 (0.8), despite the relatively high SPAD in isogenic lines 10604-5 and 10709-5 (NlLs).

Compared with RTx7000 and other Stg QTLs, Stg1 exhibited a greener leaf (higher SPAD value) and a higher WUE (based on an index calculated by the Liquor software) [figure 28].

EXAMPLE 16 The TE increased by the introgression of Stg1, may be due to a) proportionally greater photosynthetic capacity in comparison with stomatal conductance, due to smaller, thinner and greener leaves, or b) a decrease in transpiration per leaf area, keeping the At the same time, the biomass per leaf area The transpiration efficiency (TE) was negatively correlated with the leaf area transpiration (T / LA) in low and high VPD conditions (figure 29) in a set of Stl NlLs, including the recurrent parent (RTx7000). However, the classification of the Stg NlLs relative to RTx7000 interacted with the VPD conditions. For example, T / LA in Stg 1 was lower relative to RTx7000 in conditions of high VPD, even higher than RTx7000 in low VPD conditions.

In conditions of high VPD, the slope of the negative correlation between T / LA and TE was pronounced, so that a slight decrease in T / LA from 2.9 mm / cm2 (Stg4) to 2.6 mm / cm2 (Stg1) resulted a significant increase in TE from 4.2 g / m2 / mm (Stg4) to 5.1 g / m2 / mm (Stg1) [figure 29]. The gradient was less pronounced in low VPD conditions, so a six-fold decrease in T / LA was required per unit increase in TE, compared to high VPD conditions (1.2 vs 0.2 units). It should be noted that for an equivalent TE, Stg1 exhibited a T / LA greater than RTx7000 (5.1 vs. 4.6 mm / cm2) in a low VPD. This can provide a mechanism for the Stg 1 sheets to remain colder under certain environmental conditions.

EXAMPLE 17 The changes in transpiration per unit leaf area may be due to a) number of stomata, b) opening of stomata, c) changes in the synchronization of the opening and closing of the stomach relative to the VPD, and / or d) number of stomata. ciliated base cells (which affects the boundary layer and therefore TLA).

The introgression of Stg 1 in RTx7000 affected T / LA in different ways, depending on the VPD conditions. Relative to RTx7000, Stg1 increased T / LA by ~ 9% in low VPD, and decreased T / LA by ~ 10% in high VPD. Among other things, T / LA, can be regulated by a) the number of stomata per unitary leaf area, b) the size of the stomatal opening, c) the synchronization of the opening and stomatal closure, and / or d) the number of cells of ciliated base (which affects the limit layer and therefore T / LA). The measurements of two of these four components (a and d) have been made. In an experiment to protect against rain, individual leaves of the high density treatment were collected within the irrigated control, the cuticles were removed, and the images of the cuticle surface were taken. These images were used to determine a) the number of stomata per unit leaf area, b) the number of epidermal cells per unit leaf area, and c) the number of cells of ciliated base per unit leaf area.

At the same time, sections of the transverse sheet were taken. Preliminary analyzes of these data indicate that the introgression of Stg1 in RTx7000 modified the anatomy of the leaf. The differences in the morphology of the leaves (for example, Sheets 7 and 10) can be seen between RTx7000 and Stg1. In this case, there were more small packets and envelopes surrounding the vascular package in Stg1. The number of cells increased in the package envelope, should also contribute to increased photosynthetic assimilation (PNAS 2007), and therefore TE.

EXAMPLE 18 The use of increased water during grain filling is achieved through (i) increased water availability at flowering and (ii) increased water availability (better water extraction and deeper or greater lateral dispersion) during grain filling at ) Increased water availability in flowering The use of culture water (CWU) before flowering was negatively correlated with CWU after flowering in the ROS experiment (figure 30). For example, saving 20 mm of water before flowering (165 vs 185 mm) enabled the use of an additional 20 mm after the flowering (80 mm vs 60 mm). Therefore, all the water preserved before flowering was used by the crop after flowering. In general, a 25% increase in water use after flowering in this experiment resulted in a 25% increase in grain production (400 vs 300 g / m2). This resulted in 50 kg / ha of grain for each additional mm of available water. Although these data support the concept that the use of increased water is achieved during the filling of the grain by increased water availability at flowering, it does not explain with respect to the increased water access capacity during grain filling. b) Increased water accessibility during grain filling Stg1 exhibited increased water use during the period of grain filling in both low and high density treatments in the ROS experiment. This was mainly due to (i) increased water availability in flowering in high density, and (ii) increased water accessibility during low density grain filling (Figures 31A and B).

In a study of RTx7000 and four NGLs Stg (Stg1, Stg2, Stg3 and Stg4), CWU before and after flowering was negatively correlated in a ROS experiment under low density conditions (Figure 32). In this case, the Saving ~ 25 mm of water before flowering (168 vs 191 mm), contributed to the use of -50 mm after flowering (135 mm vs. 86 mm), indicating that both the availability (~ 25 mm) and the Increased access capacity (~ 25 mm) of water were equally important. However, Stg1 was anomalous in this example, since a high-level water use after flowering was associated with high-level water use before flowering. The explanations for this anomaly in Stg1 are a) an error in the pre-flowering water data, b) an error in the post-flowering water data, or c) no errors in the water measurement (Stg1 simply responded in different from other NlLs). A review of the biomass data reveals that, for some reason, Stg1 produced higher biomass before flowering in LWLD compared to the other NlLs, suggesting that water use patterns prior to flowering simply reflected a production of biomass in this experiment. However, this example does not provide evidence of the water accessibility increased by Stg1 during the period of grain filling.

EXAMPLE 19 Union between pre- and post-flowering biomass production At low density (LD), the reduction of pre-flowering biomass by 23% (from 700 to 640 g / m2) increased the post-flowering biomass by more than double (from ~ 200 to 425 g / m2).

In LD, Stg1 produced a pre-flaring biomass similar to RTx7000 (~610 g / m2), producing even less post-flow biomass (265 vs. 327 g / m2). However, in HD, Stg 1 and RTx7000 produced a similar pre-flowering biomass (~ 840 g / m2), with Stg 1 producing even more post-flowering biomass (195 vs 17 g / m2).

The relationship between GLAA and the proportion of biomass pre: post flowering is important in the history of Stg1. GLAA must be cut back to < 3 to ensure adequate water availability for grain filling, and this is an important performance of the Stg1 gene. In this experiment, Stg1 reduced GLAA in a suitable manner to achieve a pre-bloom: < 3 in LD, but not in HD. In HD, it should be noted that the introgression of Stg1 in RTx7000 reduced GLAA from 31200 to 29300 cm2 / m2, reducing the pre-flowering biomass ratio from 8.2 to 6.5 (but not yet to < 3). This indicates the importance of appropriate management strategies such as crop density in maximizing limited water resources.

The negative relationship between GLAA and stem mass after flowering is also important for the history of Stg1. The lower GLAA, and therefore the use of reduced water in flowering, was associated with a greater stem mass after flowering (a component of resistance to fixation). The introgression of Stg1 in RTx7000 increased the stem mass after flowering, in conditions of both LD (marginal increment) and HD (significant increase).

The relationship between the proportion of pre-flowering biomass (PPBR) and post-flowering biomass is instructive. The two density treatments provide continuity in the PPBR range of < 2 a > 8. When reducing PPBR from > 8 to -3, a gradual increase in the post-flowering biomass results. However, further reduction of PPBR below 3 resulted in a relatively pronounced increase in post-flowering biomass, presumably because more water was available during grain filling when the PPBR ratio fell below 3. Introgression of Stg 1 in RTx7000 increased the biomass after flowering under HD but not LD.

The relationship between the proportion of pre-flowering biomass (PPBR) and post-flowering stem mass is equally instructive. The post-flowering stem mass is a component of the resistance to fixation. The analysis of this component provides some understanding of how Stg introgressions affect fixation resistance. By reducing PPBR from > 8 a ~ 4, a gradual increase in the post flowering stem mass is obtained as a result. However, by further reducing PPBR below 4, it results in a relatively pronounced increase in post flowering stem mass. The introgression of Stg1 in RTx7000 increased post-flowering stem mass, under conditions LD (marginal increment) as HD (significant increase).

The relationship between PPBR and grain production was less clear in this experiment. Although grain production was greater in Stg1 than RTx7000 under both densities, higher production can be explained only by the PPBR lower in Stg1 in HD conditions.

Two Stg1 introgressions were reviewed in this experiment. a) 6078-1 (the entire Stg1 region), and b) 10946-5 (a recombinant coat of about 1/3 of the Stg1 region between the Sb03QGM116 and Sb03QGM106 markers).

The reduction of pre-flowering biomass by 20% (from 920 to 735 g / m2) increased the post-flowering biomass by approximately 100% (from 200 to 400 g / m2) [figure 40]. In general, the pre-flowering biomass in RTx7000 = Stg2 > Stg3 = Stg4 > Stg1 and post flowering biomass in Stg1 > Stg3 = Stg4 > Stg2 RTx7000.

The size of the cover, as is clear by GLAA, determined to a large extent the proportion of biomass pre: post flowering (figure 34). In treatments of both high and low density, the introgression of Stg1 into a background RTx7000 reduced GLAA, which in turn, reduced the ratio of biomass pre: post flowering to < 3, ensuring an adequate availability of water for filling grain under these conditions with water limitation.

The size of the cover, as is clear from GLAA, was a determinant of post flowering stem mass (PASM) [figure 35]. Under treatments of both low and high density, the introgression of Stg1 or Stgla into an RTx7000 background reduced GLAA, which in turn, increased PASM. Approximately 40 g / m2 plus stem reserves were used in HD compared to LD, reflecting a higher stress imposed by this treatment.

The relationship between the proportion of pre-flowering biomass (PPBR) and post-flowering biomass (PAB) was strong (figure 36). The two density treatments provide continuity within the PPBR range of < 1.5 a > 4. By reducing PPBR from ~ 4.5 to ~ 3.5, there was no impact on PAB. However, the further reduction of PPBR below ~ 3.5 resulted in a relatively pronounced increase in PAB, presumably because no more water is available during grain filling when the PPBR ratio falls below 3.5. The introgression of Stg1 in RTx7000 decreased PPBR below 3 in HD and LD, thus increasing PAB in both density treatments.

The proportion of pre-bloom biomass (PPBR) also affected the resistance to fixation (figure 37). In this case post-flowering stem mass (PASM) was used as a substitute for fixation resistance. At high and low densities, PPBR was negatively correlated with post-flowering stem biomass. That is, a high proportion of biomass pre: post flowering increased the quantity of reserves of remobilized stems during the filling of the grain, thus reducing the biomass of the stem and increasing the probability of fixation. In comparison with RTx7000, Stg1 significantly reduced the amount of stem reserves mobilized in LD (~ 5 vs 65 g / m2) and HD (-80 vs 140 g / m2). Compared with RTx7000, Stg 1 significantly reduced the amount of stem reserves mobilized in HD (~ 80 vs 140 g / m2), but not LD. The degree of mobilized stem reserves was higher in HD than in LD, reflecting a greater water deficit in HD. For example, the difference in mobilization of stem reserve between HD and LD was double that in RTx7000 (approximately 140 vs 70 g / m2).

Grain production remained low (in a comparative measure of ~ 4.2 t / ha) until the proportion of biomass pre: post flowering fell below ~ 3 (HD) or ~ 2.5 (LD) [figure 38]. Below these critical values, grain production increased significantly in each reduction in increment in these proportions, with the range of increase in grain production being slightly higher in LD than in HD. This suggests that the availability of water after flowering was closely related to the GLAA and pre-flowering biomass, and that a certain reduction in GLAA was required to ensure adequate water availability for the filling of the grain. Under both densities, Stg1 reduced the proportion of biomass pre: post flowering below the critical levels, resulting in increases in the production of 28% (LD) and 22% (HD), relative to RTx7000. These data provide a critical link between the action of the Stg1 gene (cover size reduced in flowering) and the production of grain under a terminal wilt. It should be noted that Stgla's introgression had some impact on the reduction of PPBR (HD and LD) and increased grain production (LD only) relative to RTx7000, but not to the same extent as Stg1. Therefore, there is no strong evidence from this experiment that the key Stg1 gene resides within the Stgla region. This supports the evidence presented above, since the strongest candidate for Stg1 (SbPIN4) is located above the Stgla introgression.

Four Stg1 introgressions were reviewed in this experiment: a) 6078-1 (the total Stg1 region); b) 10709-5 (a recombinant coat of about 1/3 of the Stg1 region); c) 10604-5 (a recombinant coat of about 3/4 of the Stg1 region); and d) 10568-2 (a recombinant cover almost 1/2 of the Stg1 region).

In a low density (LD), the pre-flowering biomass varied only -5% (from 522 to 552 g / m2) among the genotypes, even the biomass after flowering varied almost twice (from 173 to 313 g / m2) . This suggests that the considerable differences in the post-flowering biomass they were affected by something besides the pre-flowering biomass, for example, the differences in water access capacity. For example, 10709-5 and RTx7000 produced both -550 g / m2 of pre-flowering biomass, where recombinant Stg 1 (10709-5) produced ~ 60% more biomass after flowering.

In high density (HD), the pre- and post-flowering biomass were highly correlated negatively. The introgression of Stg1 in RTx7000 reduced the pre-flowering biomass by 9% and increased the post-flowering biomass by 23%.

The use of water for harvest (CWU) in flowering differed better between genotypes than in pre-flowering biomass. When combining the HD and LD data, the post-flowering biomass (PAB) remained low (in a comparative measurement of ~ 150 g / m2) until the CWU at flowering fell below ~ 180 mm. Below this critical value, PAB increased each incremental reduction in CWU below a level of 175 mm, with a PAB plateau at approximately 310 g / m2. Additional reductions in CWU at flowering below 175 mm did not result in an additional PAB.

The size of the cover, as evidenced by GLAA, largely determined the proportion of pre-flowering biomass (PPBR). Under the treatments of both high and low density, the introgression of Stg1 (or particular recombinants such as 10709-5) in an RTx7000 background, reduced GLAA, which in turn, reduced the pre-flowering biomass ratio, thus increasing the availability of water for filling the grain under these conditions with water limitation. The PPBR value for 6078-1 seems to be anomalous (too high) since this genotype is placed well above the GLAA / PPBR regression line.

The size of the cover, as evidenced by GLAA, was a determinant of post flowering stem mass (PASM). Under the high density treatment, the introgression of Stg1 (or Stg1 recombinants, such as 10604-5 and 10709-5) into an RTx7000 background, reduced GLAA, which in turn, increased PASM. Approximately 60 g / m2 more stem reserves were used in HD, compared to LD, which reflects a higher stress imposed by this treatment.

The relationship between the proportion of pre-flowering biomass (PPBR) and post-flowering biomass (PAB) was strong. The two density treatments provide continuity in the PPBR range of < 2 a > 5, although the regression slope was higher for LD than for HD. In HD, the PPBR reduction of ~6 to -3.5 resulted in a gradual increase in PAB from -130 g / m2 (RTx7000) to -180 g / m2 (10604-5). By further reducing PPBR below -3 in LD, a more pronounced increase in PAB was obtained, presumably because more water was available during grain filling when the PPBR ratio fell below 3.

The ratio of pre-bloom biomass (PPBR) also affected the resistance to fixation. In this case, the post flowering stem mass (PASM) was used as a substitute for the resistance to fixation. At both high and low densities, PPBR correlated negatively with post-flowering stem biomass. That is, a high proportion of biomass pre: post flowering, increased the amount of stem reserves remobilized during the filling of the grain, thus reducing a biomass of the stem and increasing the probability of fixation. Compared with RTx7000, Stg 1 significantly reduced the amount of stem reserves mobilized in HD (-100 vs 160 g / m2). The degree of mobilized stem reserves was higher in HD than in LD, which reflects a greater water deficit in HD. For example, the difference in mobilization of stem reserve between HD and LD was more than double in RTx7000 (approximately 160 vs 60 g / m2). Interestingly, PASM increased with the decrease of PPBR in a total range of PPBR (1.5 to 6), while grain production, and to a lesser degree PAB, only increased when PPBR fell below -3. This suggests that the relatively small water savings before flowering, still had the ability to improve the resistance to fixation, although greater savings were required of water before the grain production responded.

Grain production remained low (in a comparative measurement of -3.1 t / ha) until the proportion of biomass pre: post flowering fell below ~ 3. Below this critical value, grain production increased significantly in each reduction in increment in this proportion. Since none of the Stg1 introgressions reduced the PPBR to < 3 in HD, no production benefits were considered for Stg1 in this treatment. In LD, some of Stg1 introgressions reduced PPBR below the critical level, resulting in increases in production of 12% (10568-2) and 5% (10709-5), relative to RTx7000. These data provide a critical link between the action of the Stg1 gene (reduced cover size in flowering) and a grain production under terminal wilting.

CWU during grain filling remained low (in a comparative measurement of ~ 60 mm) until the pre-flowering biomass ratio fell below -3.5 (figure 39). Below this critical value, CWU during grain filling increased significantly for each reduction in this proportion. Since none of the Stg1 introgressions reduced the PPBR to < 3.5 in HD, no increases in CWU were considered during the filling of the grain from the Stg1 recombinants in this treatment. In LD, some of the Stg1 (and RTx7000) introgressions reduced PPBR below this critical level, increasing CWU during grain filling up to ~ 80 mm, compared to the HD baseline (-60 mm).

The relationship between CWU during grain filling and grain production was positive (figure 40). However, the correlation between these parameters was relatively low (r2 = 0.23), probably due to the highly variable nature of the measurements in the water field on the ground.

EXAMPLE 20 The delayed leaf senescence (always green) is a consequence of the state of water in the upper plant during the filling of the grain (due to the use of water increased) The water status of the plant was determined in FL-2 (two leaves under the label) in the medium grain filling using two methodologies: leaf water potential (LWP) and relative water content (RWC). LWP was measured in the field with a pressure pump. After the determination of LWP in the field, a sample of the same leaf was placed on ice, and in a few minutes, an external laboratory was taken to about 300 m for the determination of RWC through standard methods.

The RWC of FL-2 was negatively correlated with the relative senescence range of the leaf at medium grain filling at both high and low densities in a set of NgLs Stg including the recurrent parent. The correlations for HD and LD were parallel, although the compensation in approximately 0.35 units of leaf senescence, that is, for a given level of RWC, that is, 70, the relative senescence ranges of the leaf were 2.1 and 2.45 for LD and HD, respectively. The introgression of the Stg1 region in RTx7000 increased RWC in a medium grain filling (FL-2) and decreased the relative senescence range of the leaf in both HD and LD, although the impact was greater in HD.

In turn, the relative senescence range of the leaf was negatively correlated with the area of green leaf at maturity (GLAM) in HD and LD (figure 49), with higher ranges of leaf senescence exhibited in HD. Stg1 produced more than twice as much GLAM as RTx7000 in HD (2106 vs. 859 cm2 / m2) and 19% more GLAM in LD (2145 vs. 1725 cm2 / m2), see also figure 42.

EXAMPLE 21 The increased resistance to fixation is a consequence of the state of water in the upper plant during grain filling (due to the use of water increased) The mass of the upper stem at maturity is a component of the resistance to fixation. RWC in the medium grain filling (FL-2) was correlated in a highly negative way with the mass of the stem at maturity, in a set of NlLs Stg grown under conditions with water limitation at two culture densities (figure 43). The introgression of Stg 1 in RTx7000, increased RWC in the medium grain filling (FL-2) and the mass of the stem in the maturity in HD and LD, with a greater impact in HD. Relative to RTx7000, Stg 1 increased the mass of the stem at maturity by 9% in LD (224 vs. 203 g / m2) and 29% in HD (286 vs. 204 g / m2). Therefore, the benefit of Stg1, in terms of resistance to fixation, increased with the level of water deficit.

The post-flowering biomass is comprised mainly of a) post-flowering stem mass (PASM), a measure of stem reserve mobilization and a component of binding resistance, and b) grain production. Grain producers require that both grain production and resistance to fixation be maximized, that is, they do not want one thing to happen at the expense of the other. Post-flow stem mass was correlated in a highly linear manner with PAB under HD and LD conditions (figure 44). Although Stg1 had little impact on PASM under moderate wilt (LD), the amount of translocated dry mass of the stem during grain filling was much lower in Stg1, compared to RTx7000 (85 vs. 139 g / m2) in the more severe wilt (HD). This resulted in a greater stem mass at maturity in Stg1 relative to RTx7000 (286 vs. 204 g / m2), which in turn must have resistance to increased fixation.

Although the correlations between PASM and PAB were high in HD and LD (Figure 43), the correlations between grain production and PAB were low in both densities (Figure 44). The introgresiones of Stg1 in RTx7000, increased the production of grain in HD and LD, although PAB was only greater in HD. It should be noted that greater grain production was achieved in Stg1 compared to RTx7000 (290 vs. 184 g / m2) in HD, with stem reserves significantly lower in Stg1 than in RTx7000 (85 vs. 139 g / m2), This indicates that the carbon demand during the filling of the grain was largely fulfilled by the current photoassimilation and stem reserves in Stg1 and RTx7000, respectively.

The potential negotiation between PASM and grain production is shown in Figure 45. In HD, Stg1 achieved the highest grain production of any introgression (290 g / m2), while maintaining stem losses at 85 g / m2. Relative to RTx7000, Stg1 optimized the negotiation between PASM and grain production. Additional reductions in the utilization of stem stock by Stg24 (38 g / m2) resulted in lower grain production (263 g / m2). In LD, Stg1 also achieved high grain production relative to RTx7000 (282 vs. 214 g / m2), while using slightly lower stem reserves (46 vs. 50 g / m2). Therefore, in both densities, Stg1 achieved a greater grain production and resistance to fixation than RTx7000.

The relationship between PASM and stem mass at maturity was relatively flat in the various Stg introgressions in HD and LD conditions, although RTx7000 fell below the regression line in both cases. For a given level of stem reserve utilization (eg, -140 g / m2 in HD or 50 g / m2 in LD), the introgression of a Stg region in RTx7000, significantly increased the mass of the stem at maturity , suggesting that some other factor (for example, stem resistance) was important in addition to the amount of stem reserves used.

The post-flowering stem mass (PASM) was highly linearly correlated with PAB in HD and LD conditions (figure 46). The correlations HD and LD were almost parallel, although the compensation in approximately 50 g / m2, that is to say, for a determined level of PAB, that is to say, 300 g / m2, PASM was -50 g / m2 lower in HD than in LD (-100 vs. -50 g / m2). This reflects the highest level of tension in the HD treatment. In HD, both Stg1 and Stgla used -80 g / m2 stem reserves compared to almost 140 g / m2 in RTx7000, producing Stg1 still -28% more PAB than Stgla for equivalent stem reserve utilization. In LD, Stgla, and a lower grade Stg1, both increased PAB relative to RTx7000. Nevertheless, although Stgla used ~ 60 g / m2 less stem reserves than RTx7000, Stg1 used ~ 20 g / m2 more stem stocks than RTx7000.

Grain production correlated positively with PAB under HD and LD conditions. In HD conditions, Stg1 exceeded RTx7000 by 24%, although Stgla was equivalent to RTx7000 in grain production. In LD, Stg1 and Stgla exceeded RTx7000 by 42% and 20%, respectively.

In this experiment, there was no negotiation between PASM and grain production, since the correlation between these parameters was positive and linear for both cultivation densities (figure 47). In HD, Stg1 exhibited the highest stem mass and grain production of all Stg introgressions, indicating that grain production and resistance to fixation are not mutually exclusive.

The relationship between PASM and stem mass at maturity was correlated positively in HD and negatively in LD. In HD, PASM and stem mass at maturity, they were significantly higher in Stg1 than in RTx7000. In LD, the mass of the stem at maturity was higher in Stg1 than in RTx7000 (314 vs. 271 g / m2), although Stg1 used more stem reserves compared to RTx7000 (87 vs. 66 g / m2). In general, Stg1 increased the mass of the stem at maturity by 22% (HD) and 16% (LD) relative to RTx7000. Likewise, Stgla used significantly less stem reserves that RTx7000, in both harvest densities.

The post-flowering stem mass (PASM) was correlated in a highly linear manner with PAB in HD and LD conditions (figure 48). The genetic variation in the utilization of post-flowering stem reserves fluctuated from -30-110 g / m2 in LD, and from ~ 100-160 g / m2 in HD, reflecting the higher level of tension in HD treatment. In HD, the progenitor Stg1 (6078-1) and two of the recombinants (10604-5 and 10709-5), used significantly fewer stem stocks compared to RTx7000 (-100 vs. 160 g / m2), and still produced more PAB than RTx7000 (-170 vs. 130 g / m2). In LD, only one of the recombinants Stg1 (10568-2) used significantly less stem reserves than RTx7000 (-30 vs. 60 g / m2), and produced more PAB than RTx7000 (-310 vs. 230 g / m2).

Grain production was correlated positively with PAB in LD and negatively in HD, although in general (combining HD and LD), the relationship was positive, with RTx7000 (HD) as an abnormality.

In this experiment, there was no negotiation between PASM and grain production, since the correlation between these parameters was positive and linear for both cultivation densities. In HD, the progenitor Stg1 (6078-1) .exhibited a stem mass and high level grain production compared to other Stg1 injuries RTx7000 was an anomaly in HD, exhibiting a low stem mass and high grain production. In LD, 10709-5 and 10558-2 exhibited a stem mass and high level grain production relative to RTx7000.

The relationship between PASM and stem mass at maturity was correlated positively in both densities. In HD, PASM and stem mass at maturity, they were both significantly higher at 6078-1 and 10709-5 than at RTx7000. In LD, only one recombinant Stg1 (10568-2) exceeded RTx7000 in PASM and the stem mass at maturity.

EXAMPLE 22 The greater production of grain is a consequence of a state of water in the greater plant during the filling of grain The introgression of Stg1 into an RTx7000 background increased the water status of the plant in the medium grain filling, as indicated by a) higher relative water content (RWC) in FL-2 in LD and HD, lower potential of water in the sheet (LWP) in FL-2 in LD and HD. In general, the plants had more tension in LD than in HD in this experiment, evidenced by RWC in LD. However, the beneficial impact of Stg1 on the water status of the plant was more dramatic in HD, where RWC was 26% higher in Stg1 than RTx7000.

RWC in a medium grain filling in FL-2, was correlated in a positive way with the grain production in HD and LD. At higher levels of water stress in the plant (RWC <73), grain production was higher in LD than in HD, for a given level of RWC. RWC and grain production was higher in Stg1 than in RTx7000 in both cultivation densities. For example, in Stg1, under HD, a 26% increase in RWC was associated with a 58% increase in grain production, relative to RTx7000.

The leaf water potential (LWP) of FL-2 in the medium grain filling was negatively correlated with the grain production in HD and LD conditions (figure 49). Under both densities, Stg1 exhibited a lower LWP. (less tension) and higher grain production relative to RTx7000. These data, together with the RWC data, provide strong evidence of a link between the water status of the upper plant during grain filling, and increased grain production due to the Stg 1 region.

EXAMPLE 23 Increased grain production and larger grain size are consequences of water availability increased during the filling of the grain Important for the hypothesis of the function of Stg1, is the relation between the use of pre and post flowering water, and Subsequently, the relationship between the use of water development and grain production. The Stg1 gene is of little value at the field level, where there is no relationship between increased water availability during grain filling and either production or grain size.

First, it is important to establish the union between the ratio of pre-bloom biomass (PPBR) and the use of crop water (CWU) during grain filling. CWU during grain filling remained low (in a comparative measurement of ~ 85 and 95 mm for LD and HD, respectively) until the proportion of biomass pre: post flowering fell below ~ 3 and 2.5 in LD and HD, respectively ( figure 50). Below this critical value, CWU during grain filling increased significantly for each increased reduction in this proportion. Under both densities, Stg1 introgressions reduced PPBR enough to significantly increase CWU relative to RTX7000.

Second, it is important to show the union between CWU during grain filling and grain production. In general, these parameters were positively associated in an ROS experiment, in addition to two different abnormalities in LD (Stg2 and Stg3) [figure 51]. The introgression of Stg1 in RTx7000 increased both CWU during grain filling and grain production under both densities of culture.

Finally, the union between PPBR and the production of grain in conditions with water limitation completes the illustration (figure 51). Grain production remained low (in a comparative measurement of ~ 410 g / m2) until the proportion of biomass pre: post flowering fell below -2.7 (figure 52). Below this critical value, grain production increased significantly for each incremental reduction in this proportion. Under both densities, Stg1 introgressions reduced PPBR enough to significantly increase grain production relative to RTx7000. These data provide a critical link between the action of the Stg1 gene (reduced cover size in flowering) and the production of grain under terminal wilt.

CWU during grain filling was correlated positively with grain size in both HD and LD treatments (figure 53). The introgression of Stg1 into RTx7000 significantly increased the grain size in HD, but not in LD.

The relationship between PPBR and grain size under water-limited conditions indicates the importance of preserving water before flowering as a determinant of grain size (Figure 54). The grain size remained low (in a comparative measurement of ~ 17 and 17.5 mg for HD and LD, respectively) until the proportion of Pre biomass: post flowering fell below ~ 2.7 (figure 54). Below this critical value, the grain size increased significantly for each reduction in increment in this proportion. In HD but not in LD, Stg1 introgressions reduced PPBR enough to significantly increase grain size relative to RTx7000. These data provide a critical link between the action of the Stg1 gene (reduced cover size in flowering) and grain size in terminal wilt.

CWU during grain filling remained low (in a comparative measurement of ~ 58 mm for HD) until the pre-flowering biomass ratio fell below ~ 3.5. It should be noted that PPBR did not fall below the critical threshold value in any HD genotype, since CWU during grain filling remained relatively low for all genotypes in this treatment. However, as genotypes fell below this critical value in LD treatment, CWU during grain filling increased significantly for each incremental reduction in this proportion. Only one recombinant Stg1 (10709-5) increased CWU during grain filling relative to RTX7000.

In general, CWU during grain filling and grain production was correlated positively using a combined data set of treatments HD and LD, with genotypes that use more water and produce more grain in LD. However, the relationship between these parameters was not very strong. Neither the particular Stg1 recombinants consistently exceeded RTx7000 in HD and LD treatments. See figure 55.

EXAMPLE 24 Analysis of QTL gene and PIN QTL analysis We collected always green QTL data from 7 studies (Crasta and associates, 1999, Hausmann and associates, 2002, Kebede and associates, Theoretical and Applied Genetics 103: 266 to 276, 2001, Srininvas and associates, Theor Appl Genet 118: 703 to 717 , 2009, Subudhi and Associates, Theor App Genet 101: 733-741, 2000, Tao and Associates, Theor Appl Genet 100: 1225-1232, 2000, Xu and Associates, Genome 43: 461-469, 2000). Of the 7 studies, 47 individual QTLs were identified and projected onto the sorghum consensus map (Mace and associates, BMC Plant Biol. 9: 13, 2009). When Reliability Intervals (Cl) of QTL were estimated for the same overlapped feature, the QTLs were grouped into a meta QTL. Nine meta-QTL for the always green trait were identified in this way. QTL for the same trait was classified as a separate QTL, if Cl did not have a common region and the average QTL location was less than or equal to 15cM outside of each other.

The QTL analysis of Machine Learning Statistics (SML) (Bedo and associates, BMC Genetics 9: 35, 2008), was carried out in a set of approximately 500 entries in the male trait of male sorghum DEEDI. 23 QTL identified with a probability of < 0.05, were also plotted on the consensus map.

Gene analysis PIN All PIN genes available in rice and Arabidopsis were searched through NCBI (www.ncbi.org). In total, the sequence of 9 rice PIN genes (OsPINI, OsPINIb, OsPINIc, OsPIN2, OsPIN3a, OsPIN3b, OsPIN4, OsPIN5, OsPIN6) and 3 PIN genes Arabidopsis (AtPIN, AtPIN2, AtPIN4), were identified. All the genes (protein sequence) were BLASTed against sorghum WGS (www.gramene.org) and the 100 major hits were identified. The qualification (S value: a measure of similarity of the query in the sequence shown), the E value (the probability due to the opportunity, which is another alignment with a similarity higher than the determined S rating),% ID and the length of sequence homology for each of the 1200 hits. The relationship between 4 measures was analyzed, and the S score was selected as the main measure to evaluate the likelihood of sequence similarity. After an analysis of the distribution of the rating values S, three rating categories S, (> 1000; > 499 &< 1000; < 499) and a list of 11 (Table 8) were identified. sorghum genes with ratings > 499 (for example, in the first 2 categories).

Table 8 Comparisons Of the 11 PIN orthologs identified, 10 (90.9%) were aligned with known QTLs for the always green trait. Only one of the 11 pine sorghum genes (Sb03g043960 in SBI-03, did not align with any reported QTL.) Of the 79 traced QTL (23 SML-QTL and 56 literature / meta-QTL), 30 (38%) They lined up with the PIN orthologs.

EXAMPLE 25 Analysis of PIN2, PIN3, PIN4 and PIN5 The always green source BTx642 (B35), and almost isogenic lines (NlLs) containing the QTLs Stg1, Stg2, Stg4, as well as the contrasting senescence line of Tx7000, were grown in root lines in a greenhouse.

The purpose of the experiment was to measure the expression levels of the genes that are identified in the present invention as candidates for the always green gene under irrigation conditions, and after a wilt tension has been imposed on the plants, to see if there were some differences in expression in the always green trait compared to the senescent plants.

The experiment was divided into two parts: an early wilting tension (Exp1) and a late wilting tension (Exp2).

The results of the early wilt tension experiment are shown in Table 9.

Table 9 The main differences in the expression of these PIN genes are summarized below (Table 10 and Figures 67A to D).

Table 10 Emerging patterns were identified for SbPIN4 and SbPIN2, Stg1 and Stg2, respectively.

In both cases, the expression of these genes was higher in the green lines compared to the senescence line in response to water deficit.

These two PIN genes showed differences in tissue specificity. SbPIN4 generally (in all conditions) was more highly expressed in roots and stems, and less in leaves, whereas SbPIN2 generally showed greater expression in leaves and stems, and lower expression in roots).

Those skilled in the art will appreciate that the aspects described herein are susceptible to variations and modifications in addition to those specifically described. It will be understood that these aspects include all variations and modifications. The aspects described herein also include all steps, features, compositions and compounds referred to or indicated in the present invention, individually or collectively, and any and all combinations of any two or more of the steps or features.

BIBLIOGRAPHY Andrade and associates, Crop Sci. 42: 1173 to 1179, 2002 Bedo and associates, BMC Genetics 9: 35, 2008 Benkova et al., Cell 115: 591 to 602, 2003 Borrell and associates, Crop Sci. 40: 1026-1037, 2000a Borrell and associates, Crop Sci. 40: 1037-1048, 2000b Borrell and Hammer, Crop Sci. 40: 1295-1307, 2000 Carraro et al., Plant Physiology 142: 254-264, 2006 Christopher and associates, Aust. J. Agrie. Res. 59: 354 to 364, 2008 Crasta and associates, Molecular and General Genetics 262: 579 to 588.1999 Forestan and Varotto, Plant Physiology, 2009 Friml and associates, Current Opinion in Plant Biology 6: 7 to 12, 2003 Garud and associates, Int. Sorghum and Millets Newsl. 43: 63-65, 2002 Hammer and associates, Aust. J. Agrie. Res. 48: 649 to 655, 1997 Hammer, Agrie. Sci. 19: 16 to 22, 2006 Harris and Associates, J. Exp. Bot. 58: 327-338, 2007 Hausmann and Associates, Theoretical and Applied Genetics 106: 133-142, 2002 Henderson and associates, Aust. J. Plant Physiol. 25: 111-123, 1998 Henzell and Associates, Australia Int. Sorghum and Millets Newsl. 55: 1-9, 1997 Jiao and associates, Nature Genetics 42: 541 to 544, 2010 Jordán and associates, Theor. Appl. Genet 706: 559 to 567, 2003 Kashiwagi et al., Plant Physiology and Biochemistry istry 44: 152 to 157, 2006 Kassa un and associates, Euphytica 72: 351 to 362, 2010 Kebede and Associates, Theoretical and Applied Genetics 103: 266-276, 2001 Leyser, Current Biology 76: R4242 to R433, 2001 Mace and associates, BMC Plant Biol 9: 13, 2009 Manschadi and associates, Funct. Plant. Biol. 33: 823 to 837, 2006 Miura et al., Nature Genetics 42: 545-549, 2010 Mortlock and Hammer, J. Crop Prod. 2: 265-286, 1999 Papanov and associates, Trends in Plant Science 10 (4): 170-177, 2005 Passioura, J. Aust. Inst. Agrie. Sci. 43: 117 to 120, 1977 Rashotte and Associates, Plant Cell 13: 1683-1697, 2000 Reddy and Associates, Euphytica 159: 191-198, 2008 Reinhardt and associates, Plant Cell 12: 507-518, 2000 Rosenow and associates, Agrie. Water Manag. 7: 207-222, 1983 Sadras and Connor, Field Crops Res. 26: 227-239, 1991 Spano and associates, J. Exp. Bot. 54: 1415 to 1420, 2003 Springer, Nature Genetics 42: 475 to 476, 2010 Srininvas and Associates, Theor Appl Genet 118: 703-717, 2009 Subudhi and associates, Theor Appl Genet 101: 733-741, 2000 Tao and associates, Theor Appl Genet 100: 1225 to 1232, 2000 Tenkouano and associates, Theor. Appl. Genet 85: 644-648, 1993 Turner, J. Exp. Bot. 55: 2413 to 2425, 2004 Van Oosterom and Associates, Field Crops Res. 115: 19-28, 2010a Van Oosterom and associates, Field Crops Res. 115: 29-38, 2010b Van Oosterom and Hammer, Field Crops Res. 108: 259-268, 2008 Wang and associates. Molecular Plant 2 (4): 823-831, 2009 Xin and Associates, Field Crops Res. 111: 74-80, 2009 Xu and associates, Genome 43: 461 to 469, 2000 Zheng and associates, Plant Breed 128: 54-62, 2009.

Claims (24)

1. A method for generating a genetically modified plant that uses water more efficiently than a non-genetically modified plant of the same species, wherein the method comprises modulating the expression level of an existing pinhole inflorescence locus (PIN) or introduced in all the tissue, or in selected tissues in the plant, to facilitate an ever green phenotype, where the phenotype includes a change in the use of water in the period after flowering or an increased water access capacity during the growth of the cultivation, and an increased transpiration efficiency that results in an increased harvest rate and grain production under conditions with water limitation.

2. The method as described in the claim 1, characterized in that the PIN locus encodes a PIN protein selected from the list consisting of SbPINI to SbPIN11 of sorghum, or an equivalent thereof in another plant.

3. The method as described in claim 1, characterized in that the PIN is SbPIN4 or an equivalent thereof in another plant.

4. The method as described in claim 1, characterized in that the PIN is SbPIN2 or an equivalent thereof in another plant.

5. The method as described in the claim 1, characterized in that the PIN is OsPIN5 or OsPIN3a or an equivalent thereof in another plant.

6. The method as described in claim 1, characterized in that it comprises introducing a genetic agent that encodes a PIN protein or that increases or decreases the levels of a native PIN protein.

7. The method as described in claim 1, characterized in that the genetic agent is a plant region or genome selected from a particular PIN expression profile.

8. The method as described in claim 7, characterized in that the genetic agent is introduced through genetic engineering means or through a seeding protocol.

9. The method as described in the claim 1, characterized in that the genetically modified plant is a sorghum plant.

10. The method as described in claim 1, characterized in that the genetically modified plant is selected from wheat, oats, corn, barley, rye and rice.

11. The method as described in claim 1, characterized in that the genetically modified plant is selected from abaca, alfalfa, almond, apple, asparagus, banana, bean, blackberry, broad bean, cañola, cashew, cassava, chickpea, citrus, coconut, coffee, corn, cotton, fig, flax, grapes, peanuts, hemp, kenaf, lavender, hand, mushroom, olive, onion, pea, peanut, pear, millet, potato, ramie, rapeseed, ryegras, soybean, strawberry, beet, cane sugar, sunflower, sweet potato, taro, tea, tobacco, tomato, triticale, truffle and yam.

12. The method as described in the claim 1, characterized in that the ever green phenotype also includes a selected phenotype of increased cover architecture plasticity, reduced cover size, increased biomass per unit leaf area at flowering, higher transpiration efficiency, increased water usage during filling grain, reduced biomass production pre- and post-flowering and delayed senescence.

13. The method as described in claim 1, characterized in that the ever green phenotype also includes a larger grain size.

14. A genetically modified plant generated through the method as described in any of claims 1 to 13.

15. The progeny of the plant as described in claim 14, characterized in that it is genetically modified to exhibit the ever green phenotype.

16. Seeds or fruits or other reproductive propagation parts of the plant as described in claim 14 or 15.

17. A method to genetically generate a plant modified that uses water more efficiently than a non-genetically modified plant of the same species, where the method comprises introducing into a plant or progenitor of the plant, a genetic agent that encodes a PIN protein selected from SbPINI to 11 sorghum, or a functional equivalent thereof of another plant that is associated with an ever green phenotype, wherein the phenotype includes a change in water use in the post-flowering period, or an increased water accessibility during the growth of the crop or increased transpiration efficiency which results in an increased grain collection and production rate under conditions with limited water, or an agent that modulates the expression of a native PIN locus.

18. The method as described in the claim 17, characterized in that the SbPIN protein is selected from the group consisting of SbPIN4, SbPIN2, OsPIN5 and OsPIN3a or an equivalent thereof in another plant.

19. An isolated genetic material, which, when expressed in a plant cell, confers a phenotype in the plant, where the phenotype includes a change in water use in the post-flowering period or increased water access capacity during growth of the crop or increased transpiration efficiency that results in increased grain yield and grain yield under conditions with water limitation, and wherein the genetic material encodes a protein selected from SbPINI to 11 or an equivalent thereof from another plant.

20. The isolated genetic material as described in claim 19, characterized in that the SbPIN protein is selected from SbPIN4 and SbPIN2 or an equivalent thereof in another plant.

21. The isolated genetic material as described in claim 19 or 20, characterized in that the genetic material is expressed in a genetically modified sorghum plant.

22. The genetic material isolated as described in claim 19, characterized in that the genetic material is expressed in a genetically modified plant selected from wheat, oats, corn, barley, rye and rice.

23. The isolated genetic material as described in claim 19, characterized in that the genetic material is expressed in a genetically modified plant selected from abacá, alfalfa, almond, apple, asparagus, banana, bean, blackberry, broad bean, cañola, cashew, cassava , chickpea, citrus, coconut, coffee, corn, cotton, fig, flaxseed, grapes, peanut, hemp, kenaf, lavender, hand, fungus, olive, onion, pea, peanut, pear, millet, potato, ramie, rapeseed , ryegras, soybeans, strawberries, beets, sugar cane, sunflower, sweet potato, taro, tea, tobacco, tomato, triticale, truffle and yam.

24. A model of improved economic gains in crop production, where the model comprises generating crop plants that comprise a roof architecture that facilitates a change in the water use by the plant in the post-flowering period, or a capacity of Increased water access during crop growth or increased transpiration efficiency, to thereby increase HL and grain production under water limiting conditions, obtaining a seed from the cultivated crop plant and distributing the seed to the producers of grains to produce increased yields and gains, wherein the crop plant is generated by the method as described in any of claims 1 to 13 or 17 or 18.